RU2709952C2 - Method of producing higher alcohols - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: inventions relate to biotechnology. Disclosed are a reaction mixture for producing at least one high alcohol in an aqueous medium, a method of producing at least one higher alcohol in an aqueous medium, using said reaction medium to obtain at least one higher alcohol. Reaction mixture contains a mixed culture of an acetogenic microorganism selected from Clostridium ljungdahlii and Clostridium autoethanogenum, and Clostridium kluyveri in an aqueous medium containing gaseous carbon monoxide. Method envisages production of at least one higher alcohol in an aqueous medium containing said reaction mixture.

EFFECT: inventions provide one-stage production of higher alcohols in the presence of carbon monoxide.

14 cl, 4 tbl, 10 ex

Description

FIELD OF THE INVENTION

The present invention relates to a biotechnological method for producing higher alcohols from a carbon source. In particular, the mixture and method relates to the biotechnological preparation of at least one higher alcohol in the presence of carbon monoxide.

BACKGROUND OF THE INVENTION

Higher alcohols have several uses, including those used as fuel and in the perfume and cosmetics industry. For example, hexanol is commonly used in the perfume industry.

Heptanol, for example, is used in experiments on cardiac electrophysiology to block gap junctions and increase axial resistance between myocytes. An increase in axial resistance will decrease the rate of conduction and increase the sensitivity of the heart to return excitation and persistent arrhythmias. In addition, 1-heptanol has a pleasant odor and is used in cosmetics because of its aroma.

These higher alcohols can also be used as fuel in the future and can replace gasoline in the long run. For these and other reasons, there is already a potential market for higher alcohols. Higher alcohols are also used as industrial solvents.

Currently, higher alcohols are primarily made from petroleum products. These compounds are obtained by cracking gasoline or petroleum products, which is unfavorable for the environment. In addition, since the cost of these raw materials will be related to the price of oil, with the expected increase in the price of oil products in the future, prices for higher alcohols can also increase relative to the increase in prices for oil products.

The Alfol® alcohol process is a process used to produce higher alcohols from ethylene using an organoaluminum catalyst. The reaction produces linear long chain linear primary alcohols (C ₂ -C ₂₈ ). The method uses an aluminum catalyst to oligomerize ethylene and to oxidize the resulting alkyl group. However, this method gives a wide range of alcohols and the distribution model is preserved. This constant model limits the producer’s ability to produce only a certain range of alcohols, which is most in demand or of greater economic importance. In addition, the gases needed in the reaction must be very pure, and for the reaction to succeed, a special gas composition is required.

WO2009100434 also describes an indirect method for producing butanol and hexanol from a carbohydrate. The method involves homoacetogenic fermentation to obtain an intermediate product of acetic acid, which is then chemically converted to ethanol. Then, ethanol and the remainder of the acetic acid intermediate are used as a substrate in acid-forming fermentation to give butyric and caproic acid intermediates, which are then chemically converted to butanol and hexanol. However, this method uses expensive carbohydrate raw materials, and there are two additional steps of the method, the formation of esters and chemical hydrogenation of esters, which makes the method not only longer, but also simultaneously leads to the loss of useful material.

Perez J.M. (2012) discloses a method for converting short chain carboxylic acids to their corresponding alcohols in the presence of synthesis gas using Clostridium ljungdahlii. However, short chain carboxylic acids must be added as a substrate to be converted to the corresponding higher alcohol.

Currently available methods for producing higher alcohols, therefore, have limitations in transferring the mass of gaseous substrates to the fermentation broth, low productivity and lower concentrations of the final products, which leads to increased energy consumption for product purification.

Accordingly, it is required to find more environmentally friendly raw materials in addition to pure sources based on petroleum products or corn, as starting materials for the production of higher alcohols using biotechnological means, which also do less harm to the environment. In particular, there is a need for a simple and effective biotechnological production with a single reactor of higher alcohols from environmentally sound raw materials.

DESCRIPTION OF THE INVENTION

The present invention provides a reaction mixture of at least two microorganisms in the presence of carbon monoxide (CO), where the first microorganism may be able to convert CO to acetate and / or ethanol and the second microorganism may be able to convert acetate and / or ethanol to at least one acid, and the first microorganism may then be able to convert this acid to the corresponding higher alcohol, where all steps can be carried out in the presence of CO, and the higher alcohol contains at least 6 carbon atoms or more. In particular, CO is present in the reaction mixture as a gaseous substrate and CO is present in the gaseous substrate at a concentration of 2% and / or more.

In one aspect of the present invention, there is provided a reaction mixture comprising a mixed culture of the first and second microorganisms in an aqueous medium containing gaseous carbon monoxide, where

- the first microorganism is an acetogenic microorganism capable of converting a carbon source into acetate and / or ethanol, and

a second microorganism capable of converting acetate and / or ethanol to form an acid selected from the group consisting of Clostridium kluyveri and C. carboxidivorans,

where the first microorganism is additionally able to convert the acid into the corresponding higher alcohol, where the higher alcohol contains at least 6 carbon atoms.

According to another aspect of the present invention, there is provided a method for producing at least one higher alcohol in an aqueous medium containing a culture of the first and second microorganism, wherein

- the first microorganism is an acetogenic microorganism capable of converting a carbon source containing carbon monoxide into acetate and / or ethanol, and

a second microorganism capable of converting acetate and / or ethanol to form an acid selected from the group consisting of Clostridium kluyveri and C. carboxidivorans,

where the first microorganism is additionally able to convert the acid into the corresponding higher alcohol, and the higher alcohol contains at least 6 carbon atoms.

According to another aspect of the present invention, a method for producing at least one higher alcohol in an aqueous medium comprises the steps of:

(a) adding a first acetogenic microorganism capable of converting a carbon source containing carbon monoxide to acetate and / or ethanol in an aqueous medium, and

(b) add a second microorganism capable of converting acetate and / or ethanol to form an acid selected from the group consisting of Clostridium kluyveri and C. carboxidivorans,

where the first microorganism is additionally able to convert the acid into the corresponding higher alcohol, and the higher alcohol contains at least 6 carbon atoms.

In one example, steps (a) and (b) are performed simultaneously. In another example, steps (a) and (b) are performed sequentially. The concentration of the first and second microorganism can be constantly maintained to maintain the progress of the reaction. In one example, before step (b) is performed, the concentration of ethanol and / or acetate is measured to ensure that the optimal concentration for the second microorganism is achieved. In particular, the concentration of acetate and / or ethanol may be at an optimum level so that there is enough substrate in the aqueous medium so that the second organism forms at least one higher acid. A person skilled in the art will be able to easily determine the appropriate time for incorporation of the second organism into the aqueous medium.

In another example, steps (a) and (b) are performed simultaneously. In this example, the second microorganism may be inactive (i.e., may remain latent) before a sufficient amount of acetate and / or ethanol is made available through the activity of the first microorganism. The presence of one of the microorganisms in the aquatic environment does not violate or interfere with the activity and / or effectiveness of the other.

An advantage of the present invention may be that much more favorable CO ₂ / CO mixtures of feed can be used. These various sources include natural gas, biogas, coal, oil, plant residues and the like. Another advantage of the process may be a high carbon yield. This was made possible by returning the generated CO ₂ . Namely, CO ₂ can react in the first step back to acetic acid.

Another advantage may be greater flexibility with respect to the fermentation conditions used, since any acetogenic microorganism and any microorganism capable of carrying out the ethanol-carboxylate fermentation pathway can be used in combination to actually produce higher alcohols. Another advantage of the present invention may be that since the second microorganism can function and / or produce acid from acetate and / or ethanol in the presence of CO, simultaneously the first and second microorganisms can be present in a homogeneous mixture to produce higher alcohols from a carbon source containing CO . This feature of the second microorganism allows the production of higher alcohols from a carbon source, such as CO, to be a one-step process, making this process more efficient and yield greater. Because of this, the advantages of the second microorganism, a one-step procedure for the manufacture of higher alcohols can be carried out in a single fermenter without an intermediate separation step. It is also possible to increase the concentration of the final product using this one-step procedure. Surprisingly, Baffert C., 2011 and Thauer RK, 1973 both reported that hydrogenases were inhibited in the presence of CO. For this and other reasons, WO2013 / 167663 includes the step of separating between (a) the step of forming acetate and / or ethanol from CO and / or CO ₂ in the presence of an acetogenic organism and (b) the step of forming a hydrocarbon containing at least one oxygen atom (e.g. , hexanoic acid), in the presence of a second microorganism. The ability to produce a higher alcohol, in particular one that contains at least 6 carbon atoms, in one CO synthesis reactor according to any aspect of the present invention, is thus an amazing result. In any case, even if steps (a) and (b) are carried out in two separate steps (i.e., in two separate containers), there may be no need for a specific recovery method to remove all trace amounts of CO for both the first and second microorganisms.

As can be seen in the examples, the presence of CO makes it possible to obtain hexanol in the method according to any of the aspects of the present invention, where the carbon source contains at least CO.

A carbon source containing CO can be converted into at least one acid in the presence of at least one acetogenic microorganism and a second microorganism capable of carrying out an ethanol-carboxylate fermentation pathway. In particular, an acid may contain 6 or more carbon atoms. More specifically, the formed acid can be selected from the group consisting of hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid decanoic acid and the like. In particular, a carbon source containing CO in the presence of at least one acetogenic bacterium can produce ethanol and / or acetic acid.

The expression “acetogenic bacteria” as used herein refers to a microorganism that is capable of following the Wood-Lungdal pathway and thus is capable of converting CO, CO ₂ and / or hydrogen to acetate. These microorganisms include microorganisms that, in their wild-type form, do not follow the Wood-Ljungdal pathway, but acquired this trait as a result of genetic modification. Such microorganisms include, but are not limited to, E. coli cells. These microorganisms may also be known as carboxidotrophic bacteria. Currently, 21 genus of various acetogenic bacteria are known in the art (Drake et al., 2006), and they may also include some Clostridia (Drake & Kusel, 2005). These bacteria are able to use carbon dioxide or carbon monoxide as a carbon source with hydrogen as an energy source (Wood, 1991). In addition, alcohols, aldehydes, carboxylic acids, and also numerous hexoses can also be used as a carbon source (Drake et al., 2004). The reduction pathway that leads to the formation of acetate is called the acetyl-CoA or Wood-Lungdal path.

In particular, acetogenic bacteria can be selected from the group consisting of Acetoanaerobium notera (ATCC 35199), Acetonema longum (DSM 6540), Acetobacterium carbinolicum (DSM 2925), Acetobacterium malicum (DSM 4132), species Acetobacterium No. 446 al. Moraga , 1990, J. Biotechnol., Vol. 14, p. 187-194), Acetobacterium wieringae (DSM 1911), Acetobacterium woodii (DSM 1030), Alkalibaculum bacchi (DSM 22112), Archaeoglobus fulgidus (DSM 4304), Blautia producta ( DSM 2950, formerly Ruminococcus productus, formerly Peptostreptococcus productus), Butyribacterium methylotrophicum (DSM 3468), Clostridium aceticum (DSM 1496), Clostridium autoethanogenum (DSM 10061, DSM 19630 and DSM 23693), Clostridium DSMocidio carboxid No. PTA-10522), Clostridium drakei (ATCC BA-623), Clostridium formicoaceticum (DSM 92), Clostridium glycolicum (DSM 1288), Clostridium ljungdahlii (DSM 13528), Clostrid ium ljungdahlii C-01 (ATCC 55988), Clostridium ljungdahlii ERI-2 (ATCC 55380), Clostridium ljungdahlii O-52 (ATCC 55989), Clostridium mayombei (DSM 6539), Clostridium methoxybenzovoidm (DSM158) , Clostridium scatologenes (DSM 757), species Clostridium ATCC 29797 (Schmidt et al., 1986, Chem. Eng. Commun., Vol. 45, p. 61-73), Desulfotomaculum kuznetsovii (DSM 6115), Desulfotomaculum thermobezoicum of the subspecies thermosyntrophicum (DSM 14055), Eubacterium limosum (DSM 20543), Methanosarcina acetivorans C2A (DSM 2834), species Moorella Hai al et al. 2004 (2004). Let., Vol. 29, p. 1607-1612), Moorella thermoacetica (DSM 521, formerly Clostridium thermoaceticum), Moorella thermoautotrophica (DSM 1974), Oxobacter pfennigii (DSM 322), Sporomusa aerivorans (DSM 13326), Sporomusa o DSM 2662), Sporomusa silvacetica (DSM 10669), Sporomusa sphaeroides (DSM 2875), Sporomusa termitida (DSM 4440) and Thermoanaerobacter kivui (DSM 2030, formerly Acetogenium kivui). More specifically, the ATCC BAA-624 strain of the species Clostridium carboxidivorans can be used. Even more specifically, a bacterial strain of Clostridium carboxidivorans labeled “P7” and “P11” can be used, as described, for example, in US Patent Application No. 2007/0275447 and US Patent Application No. 2008/0057554.

Another particularly suitable bacterium may be Clostridium ljungdahlii. In particular, strains selected from the group consisting of Clostridium ljungdahlii PETC, Clostridium ljungdahlii ERI2, Clostridium ljungdahlii COL and Clostridium ljungdahlii O-52 can be used to convert synthesis gas to hexanoic acid. These strains, for example, are described in WO 98/00558, WO 00/68407, ATCC 49587, ATCC 55988 and ATCC 55989. In another example, the acetogenic bacterium selected for the first organism may be Clostridium autoethanogenum.

Acetogenic bacteria can be used in combination with a second microorganism that is capable of carrying out an ethanol-carboxylate fermentation pathway. In one example, an acetogenic bacterium and a second microorganism that is capable of carrying out an ethanol-carboxylate fermentation pathway can simultaneously be used to produce higher alcohol from a carbon source containing CO. The acid can then be converted to the corresponding higher alcohol selected from the group consisting of hexanol, octanol, nonanol, decanol and the like. In one example, the higher alcohol may be selected from the group consisting of 1-hexanol, 2-hexanol, 3-hexanol, 1-heptanol, 2-heptanol, 3-heptanol, 4-heptanol, octanol, nonanol, decanol and the like.

In one example, ethanol and / or acetate can be converted to the corresponding higher acid in the presence of a second microorganism capable of carrying out the ethanol-carboxylate fermentation pathway. The ethanol-carboxylate fermentation pathway has been described in detail at least in Seedorf H. et al., 2008. In particular, the second microorganism may be selected from the group consisting of Clostridium kluyveri, C. Carboxidivorans and the like. These second microorganisms include microorganisms that, in their wild-type form, do not carry out the ethanol-carboxylate fermentation pathway, but have acquired this trait as a result of genetic modification. In particular, the second microorganism may be Clostridium kluyveri.

In another example, the second microorganism may be a wild-type organism that expresses at least one enzyme selected from the group consisting of E₁ - E_elevenwhere E₁ represents alcohol dehydrogenase (adh), E₂ represents acetaldehyde dehydrogenase (ald), E₃ represents acetoacetyl-CoA-thiolase (thl), E₄ represents 3-hydroxybutyryl-CoA-dehydrogenase (hbd), E₅ represents 3-hydroxybutyryl-CoA-dehydratase (crt), E₆ represents butyryl-CoA-dehydrogenase (bcd), E₇ is a subunit of electron-transporting flavoprotein (etf), E₈ Coenzyme A transferase (cat), E₉ represents acetate kinase (ack), E₁₀ represents phosphotransacetylase (pta) and E_eleven is a transhydrogenase. In particular, a second wild-type microorganism according to any aspect of the present invention may express at least E₂, E₃ and E₄. Even more specifically, a second wild-type microorganism according to any aspect of the present invention can express at least E₄.

In another example, the second microorganism according to any aspect of the present invention may be a genetically modified organism that has increased expression compared to the wild-type microorganism of at least one enzyme selected from E₁-E_elevenwhere E₁ represents alcohol dehydrogenase (adh), E₂ represents acetaldehyde dehydrogenase (ald), E₃ represents acetoacetyl-CoA-thiolase (thl), E₄ represents-3-hydroxybutyryl-CoA-dehydrogenase (hbd), E₅ represents 3-hydroxybutyryl-CoA-dehydratase (crt), E₆ represents butyryl-CoA-dehydrogenase (bcd), E₇ is a subunit of electron-transporting flavoprotein (etf), E₈ Coenzyme A transferase (cat), E₉ represents acetate kinase (ack), E₁₀ represents phosphotransacetylase (pta) and E_eleven is a transhydrogenase. In particular, a genetically modified second microorganism according to any aspect of the present invention can express at least E enzymes₂, E₃ and E₄. Even more specifically, a genetically modified second microorganism according to any aspect of the present invention can express at least E₄. Enzymes E₁ - E_eleven can be isolated from Clostridium kluyveri. One of skill in the art can measure the activity of each of these enzymes using methods known in the art. In particular, the activity of enzymes E₁ and E₂can be measured using analyzes reported at least in Hillmer P., 1972, Lurz R., 1979; enzyme activity E₂can also be measured using the analysis reported in Smith L.T., 1980; enzyme activity E₃ and E₄can be measured using analyzes reported at least in Sliwkowski M.X., 1984; activity E₄ can also be measured using the analysis reported in Madan V.K., 1972; activity E₅ can also be measured using the analysis reported in Bartsch R.G., 1961; enzyme activity E₆ and E₇can be measured using the analysis reported in Li F., 2008; activity E₇ can also be measured using the analysis reported in Chowdhury, 2013; activity E₈ can be measured using analysis reported in Stadman, 1953; activity E₉ can be measured using analysis reported in Winzer K., 1997; activity E₁₀ can be measured using the analysis reported in Smith L.T., 1976; and activity E_eleven can be measured using the analysis reported in Wang S., 2010.

According to any aspect of the present invention, the first and / or second microorganism may be a genetically modified microorganism. A genetically modified cell or microorganism may be genetically different from a wild-type cell or microorganism. A genetic difference between a genetically modified microorganism according to any of the aspects of the present invention and a wild-type microorganism may consist in the presence of a complete gene, amino acid, nucleotide, etc. in a genetically modified microorganism, which may be absent in the wild-type microorganism. In one example, a genetically modified microorganism according to any aspect of the present invention may contain enzymes that allow the microorganism to produce at least one carboxylic acid. A wild-type microorganism, as compared to a genetically modified microorganism according to any aspect of the present invention, may have no or undetectable enzyme activity that allows the genetically modified microorganism to produce at least one carboxylic acid. The expression ‘genetically modified microorganism’ used herein can be used interchangeably with the expression ‘genetically modified cell’. Genetic modification according to any aspect of the present invention can be carried out in a cell of a microorganism.

The phrase “wild type” used herein in combination with a cell or microorganism can refer to a cell with a genome structure that is in a form naturally occurring in the wild. The expression can be applied both to the whole cell, and to individual genes. The expression “wild type”, therefore, does not include such cells or genes where the gene sequences have been altered, at least in part, by humans using recombinant methods.

One skilled in the art will be able to apply any method known in the art for the genetic modification of a cell or microorganism. According to any aspect of the present invention, a genetically modified cell can be genetically modified in such a way that in a certain time interval, within 2 hours, in particular within 8 hours or 24 hours, it forms at least twice, preferably, according to at least 10 times, at least 100 times, at least 1000 times, or at least 10,000 times more carboxylic acid and / or the corresponding carboxylic acid ester than a wild-type cell. The increase in product formation can be determined, for example, by culturing a cell according to any aspect of the present invention and a wild-type cell, each separately, under the same conditions (the same cell density, the same culture medium, the same cultivation conditions) for a certain period of time in a suitable culture medium , and then determining the amount of the target product (carboxylic acid) in a nutrient medium.

In another example, the acid can be obtained from a carbon source containing CO by any method disclosed in Steinbusch, 2011, Zhang, 2013, Van Eerten-Jansen MCA A, 2013, Ding H. et al., 2010, Barker HA, 1949, Stadtman ER, 1950, Bornstein BT et al., 1948, etc. Even more specifically, an acid can be obtained from a carbon source containing CO in the presence of at least Clostridium kluyveri.

Even more specifically, according to any aspect of the present invention, the acid is obtained in the presence of at least one acetogenic microorganism and Clostridium kluyveri. In one example, the acetogenic microorganism may be Clostridium ljungdahlii or Clostridium ragsdahlei.

When obtaining acid from a carbon source containing CO, a combination of bacteria can be used. More than one acetogenic bacterium may be present in combination with one or more second microorganisms. In another example, an acetogenic bacterium of more than one species and a second microorganism of only one species may be present. In yet another example, more than one second microorganism may be present in combination with only one acetogenic bacterium.

The reaction mixture may contain two microorganisms in a homogeneous mixture. The term ‘homogeneous mixture’ as used herein refers to a mixture of microorganisms uniformly spatially distributed in a medium. In particular, the mixture may contain at least two microorganisms, while the acetogenic and the second microorganism are uniformly distributed in the aquatic environment. In one example, there may be approximately equal amounts of acetogenic and second microorganisms in the mixture. In another example, more acetogenic microorganism may be present in the mixture compared to the second microorganism. In yet another example, more than a second microorganism may be present in the mixture compared to an acetogenic microorganism. In all possible examples, the microorganisms are in the same homogeneous mixture, where they are evenly distributed throughout the mixture. The expression ‘aqueous medium’ used herein can be used interchangeably with the expression ‘reaction mixture’.

The expression “acetate” used in this document refers to both acetic acid and its salts, which is inevitable, because, as is known from the prior art, microorganisms carry out vital activity in the aquatic environment, and there is always a balance between salt and acid .

The expression "second microorganism" refers to a microorganism that is different from the "first microorganism" according to any aspect of the present invention.

In another example, step (a) and step (b) can be carried out in two different containers. In one example, step (a) can be carried out in a fermenter 1, where the first microorganism comes into contact with a carbon source containing CO to produce acetate and / or ethanol. Ethanol and / or acetate can then be brought into contact with the second microorganism in fermenter 2 to produce at least one acid. The acid can then be fed back to fermenter 1 to convert the acid to the desired higher alcohol. You can create a cycle where the acetate and / or ethanol obtained in fermenter 1 can be continuously fed into fermenter 2, acetate and / or ethanol in fermenter 2 can be converted into at least one acid and the acid from fermenter 2 is fed back to fermenter 1. The CO fed to fermenter 1 can be transferred to fermenter 2 along with acetate and / or ethanol. No special isolation method is needed since the second microorganism has been found to convert acetate and / or ethanol to at least one acid in the presence of CO.

In another example, the medium is recycled between fermenters 1 and 2. Thus, the ethanol and / or acetate obtained in fermenter 1 can be fed to fermenter 2, and the acid obtained in fermenter 2 can be fed back to fermenter 1. In the process of recirculating the medium CO from fermenter 1 can be introduced into fermenter 2. In addition, the acids obtained in fermenter 2 can be reintroduced into fermenter 1. The second microorganism in fermenter 2 may be able to continue to produce acids from acetate and ethanol in the presence of CO recycled from enzyme 1 and 2. Alcohols fermenter accumulated in fermenters 1 and 2, may then be isolated by means known in the art.

In a further example, three containers may be present to carry out the method according to any aspect of the present invention. The first microorganism may be present in the first fermenter, the second microorganism in the second fermenter and the third fermenter again with the first microorganism. In fermenter 1, the first microorganism comes into contact with a carbon source to produce acetate and / or ethanol. Ethanol and / or acetate can then be brought into contact with the second microorganism in fermenter 2 to produce at least one acid. The acid can then be fed to fermenter 3 to produce at least one alcohol.

In particular, CO can be fed into the aqueous medium in a continuous gas stream. The concentration of CO in the gas stream may be at least 2% by volume of the total gas in the gas stream. In particular, CO may be present in a concentration range from 2 to 99% by volume, in the range from 2 to 95% by volume, from 5 to 95% by volume, from 10 to 90% by volume, from 15 to 85% by volume in particular in the range of 20 to 80% by volume. More specifically, the concentration of CO may be approximately 7%, 24% by volume. The carbon monoxide gas phase concentration in the carbon source can be measured using at least a GC 6890N gas chromatograph from Agilent Technologies Inc. with a thermal conductivity detector.

The expression ‘approximately’ used in this document refers to changes within 20 percent. In particular, the expression “approximately” as used herein refers to +/- 20%, more specifically, +/- 10%, even more specifically, +/- 5% of a given measurement or value.

All percentages (%) are, unless otherwise indicated, volume percent.

The carbon source used according to any aspect of the present invention contains carbon dioxide and / or carbon monoxide. One skilled in the art will recognize that there are many possible sources for providing CO and / or CO ₂ as a carbon source. You can see that in practice, any gas or any gas mixture that is capable of providing microorganisms with a sufficient amount of carbon so that acetate and / or ethanol can be formed from a source of CO and / or CO can be used as a carbon source in accordance with any aspect of the present invention. ₂ .

Typically, for a mixed culture according to any aspect of the present invention, the carbon source contains at least 50% by volume, at least 70% by volume, preferably at least 90% by volume of CO and / or CO ₂ , where percent by volume -% refer to all carbon sources that are available for the first microorganism in a mixed culture.

In a mixed culture according to any aspect of the present invention, a source of carbon material may be provided. Examples of carbon sources in gaseous form include exhaust gases such as synthesis gas, flue gas, and petroleum refining gases obtained from yeast fermentation or fermentation with clostridia. These exhaust gases are produced by gasification of cellulose-containing materials or coal gasification. In one example, these exhaust gases are not necessarily obtained as by-products of other processes, but can be specifically prepared for use with a mixed culture according to any aspect of the present invention.

According to any aspect of the present invention, the carbon source may be synthesis gas. Synthesis gas can, for example, be obtained as a by-product of coal gasification. Accordingly, a microorganism of a mixed culture according to any aspect of the present invention is capable of converting a substance that is a waste product into a valuable resource. In another example, synthesis gas may be a by-product of the gasification of a widely available, inexpensive agricultural raw material for use with the mixed culture of the present invention to produce at least one higher alcohol.

There are numerous examples of raw materials that can be converted into synthesis gas, since almost all forms of vegetation can be used for this purpose. In particular, the feed is selected from the group consisting of perennial herbs, such as miscanthus, corn residues, processing waste, such as sawdust, and the like.

In general, synthesis gas can be obtained in a device for gasification of dry biomass mainly due to pyrolysis, partial oxidation and steam reforming, where the primary products of synthesis gas are CO, H ₂ and CO ₂ . Syngas can also be a product of CO ₂ electrolysis. One skilled in the art will recognize the appropriate conditions for the electrolysis of CO ₂ to produce synthesis gas containing CO in the required amount.

Typically, part of the synthesis gas obtained in the gasification process is first treated to optimize the yield of the product and prevent the formation of resins. Cracking unwanted resins and CO in the synthesis gas can be performed using lime and / or dolomite. These processes are described in detail, for example, in Reed, 1981.

Mixtures of sources can be used as a carbon source.

According to any aspect of the present invention, a reducing agent, for example hydrogen, can be supplied together with a carbon source. In particular, this hydrogen can be supplied when C and / or CO _{2 is} supplied and / or used. In one example, hydrogen gas is part of the synthesis gas present according to any aspect of the present invention. In another example, where hydrogen gas in the synthesis gas is not sufficient for the method of the present invention, additional hydrogen gas can be supplied.

One skilled in the art will recognize the other conditions necessary for implementing the method according to any aspect of the present invention. In particular, the conditions in the container (e.g., fermenter) may vary depending on the first and second microorganisms used. Changing conditions in such a way that they are suitable for the optimal functioning of microorganisms is within the knowledge of a person skilled in the art.

In one example, the method according to any aspect of the present invention may be carried out in an aqueous medium with a pH between 5 and 8, 5.5 and 7. The pressure may be between 1 and 10 bar.

In particular, the aqueous medium may contain a carbon source containing CO and / or CO ₂ . More specifically, a carbon source containing CO and / or CO ₂ is supplied to the aqueous medium in a continuous gas stream. Even more specifically, the continuous gas stream contains synthesis gas. In one example, gases are part of a single stream / stream. In another example, each gas is a separate stream / stream supplied to the aqueous medium. These gases can be separated, for example, using separate nozzles that open into the aqueous medium, frits, membranes inside the gas supply pipe to the aqueous medium, and the like.

In particular, a reaction mixture according to any aspect of the present invention (i.e., a mixture of a first microorganism — an acetogenic organism, a second microorganism and a carbon source containing carbon monoxide) can be used in any known bioreactor or fermenter to carry out any of the aspects of the present invention.

The term ‘higher alcohols’ as used herein refers to alcohols that contain from 6 to 10 carbon atoms and may be somewhat viscous or oily and have heavier fruity aromas. Higher alcohols may include, but are not limited to, hexanol, heptanol, octanol, nonanol, decanol, and the like. More specifically, a higher alcohol can be selected from the group consisting of 1-hexanol, 1-octanol, 1-heptanol, 3-methyl-1-pentanol, 4-methyl-1-hexanol, 5-methyl-1-heptanol, 4- methyl-1-pentanol, 5-methyl-1-hexanol, 6-methyl-1-heptanol and combinations thereof.

According to any aspect of the present invention, “the corresponding higher alcohol’ ”refers to alcohol with the same number of carbon atoms as the acid from which the corresponding higher alcohol is formed. For example, hexanoic acid can be converted to the corresponding alcohol, hexanol; heptanoic acid can be converted into the corresponding alcohol - heptanol; octanoic acid can be converted into the corresponding alcohol - octanol; nonanoic acid can be converted to the corresponding alcohol, nonanol; decanoic acid can be converted to the corresponding alcohol - decanol and the like.

In one example, the method according to any aspect of the present invention may result in a mixture of acids and / or higher alcohols. In another example, process parameters according to any aspect of the present invention may be adjusted to produce more than one acid and / or higher alcohol compared to the rest. For example, with the following parameters for growing a mixed culture on a complex medium (0.25 g / l NH ₄ Cl, 0.2 g / l MgSO ₄ x 7 H ₂ O, 0.31 g / l K ₂ HPO ₄ , 0.23 g / l KH ₂ PO ₄ , 2.5 g / l NaHCO ₃ , 1 g / l yeast extract, 10 g / l K-acetate, 20 g / l ethanol, 0.25 g / l L-cysteine-HCl, 1.5 mg / L FeCl ₂ x 4 H ₂ O, 70 μg / L ZnCl ₂ x 7 H ₂ O, 100 μg / L MnCl ₂ x 4 H ₂ O, 6 μg / L Boric Acid, 190 μg / L CoCl ₂ x 6 H ₂ O, 2 μg / L CuCl ₂ x 6 H ₂ O, 24 μg / L NiCl ₂ x 6 H ₂ O, 36 μg / L Na ₂ MoO ₄ x 2 H ₂ O, 3 μg / L Na ₂ SeOO ₃ x 5 H ₂ O, 4 μg / L Na ₂ WO ₄ x 2 H ₂ O, 100 μg / L Vitamin B12, 80 μg / L p-aminobenzoic acid, 20 μg / L Biotin, 200 μg / L Nicotinic acids, 100 μg / l Ca-pantothenic acid, 300 μg / l pi idoksin-HCl, 200 mg / l thiamine-HCl x H ₂ O) at 37 ° C with a pH of about 6.5 for approximately 240 hours, the concentration of butanol and / or receiving hexanol can be controlled.

Oxygen may be present in the reaction mixture according to any aspect of the present invention. Accordingly, the first and second microorganisms according to any aspect of the present invention can be grown aerobically. In particular, oxygen can be supplied to the aqueous medium according to any aspect of the present invention in a continuous gas stream. More specifically, the concentration of O ₂ in the gas stream can be represented by less than 1% by volume of the total amount of gas in the gas stream. In particular, oxygen may be present in a concentration range from 0.000005 to 2% by volume, in a range from 0.00005 to 2% by volume, from 0.0005 to 2% by volume, from 0.005 to 2% by volume, from 0.05 to 2% by volume, from 0.00005 to 1.5% by volume, from 0.0005 to 1.5% by volume, from 0.005 to 1.5% by volume, from 0.05 to 1, 5% by volume, from 0.5 to 1.5% by volume, from 0.00005 to 1% by volume, from 0.0005 to 1% by volume, from 0.005 to 1% by volume, from 0.05 to 1% by volume, from 0.5 to 1% by volume, from 0.55 to 1% by volume, from 0.60 to 1% by volume, in particular in the range from 0.60 to 1.5%, from 0.65 to 1% and from 0.70 to 1% by volume. In particular, an acetogenic microorganism is particularly suitable when the proportion of O ₂ in the gas phase / stream is approximately 0.00005, 0.0005, 0.005, 0.05, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2% by volume relative to the volume of gas in the gas stream. One skilled in the art will be able to apply any of the methods known in the art to measure the volume concentration of oxygen in a gas stream. In particular, the volume of oxygen can be measured using any method known in the art. In one example, the concentration of oxygen in the gas phase can be measured using a PreSens Precision Sensing GmbH immersion probe for trace amounts of oxygen. Oxygen concentration can be measured using quenching of fluorescence, where the degree of quenching is correlated with the partial pressure of oxygen in the gas phase. Even more specifically, the first and second microorganisms according to any aspect of the present invention are able to optimally carry out vital activities in the aquatic environment when oxygen is supplied with a gas stream with an oxygen concentration of less than 1% by volume of the total gas, approximately 0.015% by volume of the total volume gas in the gas stream supplied to the reaction mixture.

An aqueous medium according to any aspect of the present invention may contain oxygen. Oxygen can be dissolved in the medium by any method known in the art. In particular, oxygen may be present at a concentration of 0.5 mg / L. In particular, the concentration of dissolved free oxygen in the aqueous medium may be at least 0.01 mg / L. In another example, dissolved oxygen may be approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / L . In particular, the concentration of dissolved oxygen may be 0.01-0.5 mg / L, 0.01-0.4 mg / L, 0.01-0.3 mg / L, 0.01-0.1 mg / l In particular, oxygen can be supplied to the aqueous medium in a continuous gas stream. More specifically, the aqueous medium may contain oxygen and a carbon source containing CO and / or CO ₂ . More specifically, oxygen and a carbon source containing CO and / or CO ₂ are supplied to the aqueous medium in a continuous gas stream. Even more specifically, the continuous gas stream contains synthesis gas and oxygen. In one example, both gases are part of the same stream / stream. In another example, each gas is a separate stream / stream supplied to the aqueous medium. These gases can be separated, for example, using separate nozzles that open into the aqueous medium, frits, membranes inside the gas supply pipe to the aqueous medium, and the like. Oxygen may be free oxygen. According to any aspect of the present invention, the expression “free oxygen-containing reaction mixture” refers to a reaction mixture containing elemental oxygen in the form of O ₂ . O ₂ may be dissolved oxygen in the reaction mixture. In particular, dissolved oxygen may be present at a concentration of ≥ 5 ppm (0.000005% vol .; 5x10 ^-6 ). One of skill in the art can use any method known in the art to measure the concentration of dissolved oxygen. In one example, dissolved oxygen can be measured using oxygen immersion probes (type PSt6 from PreSens Precision Sensing GmbH, Regensburg, Germany).

In one example, according to any aspect of the present invention, the carbon source is synthesis gas and the carbon source may be mixed with gaseous oxygen before being introduced into the aqueous medium. This mixing step can increase the efficiency and production of higher alcohols in the reaction. The overall efficiency, alcohol performance and / or total carbon capture in the method of the present invention may depend on CO stoichiometry₂, CO, H₂ and O₂ in a continuous gas stream. The supplied continuous gas stream may be composition O₂, CO₂ and H₂. In particular, in a continuous gas stream, the concentration range is O₂ may range from 0.000005% to 1% by volume, CO / CO₂ about 10-50%, in particular 33% by volume, and H₂ will be in the range from 44% to 84%, in particular from 64 to 66.04% by volume. More specifically, the gas concentration in the continuous gas stream may be 0.15% by volume for O₂, 32% by volume for CO / CO₂ and 64% by volume for H₂. In another example, the continuous gas stream may also contain inert gases like N₂up to a concentration of N₂50% by volume.

One of skill in the art will understand that it may be necessary to control the composition and flow rates of the jets at regular intervals. Control of the composition of the stream can be achieved by changing the proportions of its constituent jets to achieve the desired or desired composition. The composition and flow rate of the mixed stream can be controlled by any means known in the art. In one example, a system is adapted to continuously monitor the flow rates and compositions of at least two jets and combine them to produce a single mixed substrate stream in a continuous gas stream of the optimal composition and means for passing the optimized substrate stream into the mixed culture according to any aspect of the present invention.

It is advisable to include O ₂ in the reaction mixture and / or the gas stream supplied to the reaction mixture, since most of the exhaust gases, including synthesis gas, contain oxygen in small or large quantities. It is difficult and expensive to remove this oxygen before using synthesis gas as a carbon source to produce higher alcohols. The method according to any of the aspects of the present invention allows to obtain at least one higher alcohol without first having to remove any trace amounts of oxygen from the carbon source. This saves time and money.

In one example, when O _{2 is} present in the reaction mixture according to any aspect of the present invention, the first microorganism, an acetogenic bacterium, may be present in two growth phases. In an example, at least one acetogenic bacteria can be in an exponential growth phase and other acetogenic bacteria can be in any other growth phase in the life cycle of an acetogenic microorganism. In particular, in accordance with any aspect of the present invention, acetogenic bacteria in an aqueous medium may contain one acetogenic bacteria in an exponential growth phase and others in a stationary phase. In the presence of oxygen without the presence of acetogenic bacteria in an exponential growth, acetogenic bacteria in the stationary phase may not be able to produce acetate and / or ethanol. This phenomenon is confirmed at least in Brioukhanov, 2006, Imlay, 2006, Lan, 2013, etc. The authors of the present invention, therefore, found that in the presence of acetogenic bacteria in exponential growth, acetogenic bacteria in any growth phase can aerobically breathe and produce acetate and / or ethanol in a greater or equal amount compared to that obtained when oxygen was not present in the reaction mixture. In one example, acetogenic bacteria in an exponential growth phase may be capable of removing free oxygen from the reaction mixture, providing suitable conditions (lack of free oxygen) for acetogenic bacteria in any growth phase for the metabolism of a carbon substrate containing CO and producing acetate and / or ethanol.

In another example, the aqueous medium may already contain acetogenic bacteria in any growth phase, in particular in the stationary phase, in the presence of a carbon source containing CO. In this example, oxygen may be present in the carbon source supplied to the aqueous medium, or in the aqueous medium itself. In the presence of oxygen, acetogenic bacteria may be inactive and may not produce acetate and / or ethanol until acetogenic bacteria are added in the exponential growth phase. In this very example, acetogenic bacteria in the exponential growth phase can be added to the aquatic environment. Inactive acetogenic bacteria already in the aquatic environment can then activate and begin to produce acetate and / or ethanol.

In a further example, acetogenic bacteria in any growth phase can be first mixed with acetogenic bacteria in an exponential growth phase and then a carbon source and / or oxygen can be added.

According to any aspect of the present invention, an exponentially growing microorganism growing in the presence of oxygen can lead to the formation of a microorganism that adapts to growth and metabolism in the presence of oxygen. In particular, the microorganism may be able to remove oxygen from the environment surrounding the microorganism. This newly acquired adaptation allows acetogenic bacteria in an exponential growth phase to release oxygen from the environment and thus produce acetate and ethanol from a carbon source. In particular, an acetogenic bacterium with a newly acquired adaptation allows bacteria to convert a carbon source containing CO into acetate and / or ethanol and newly formed acid into the corresponding higher alcohol in the presence of alcohol. A second microorganism selected from the group consisting of Clostridium kluyveri and C. Carboxidivorans can convert acetate and / or ethanol to form a newly formed acid. As mentioned earlier, it is preferable that this process is carried out in the presence of O ₂ (i.e., with the inclusion of O ₂ in the reaction mixture), since most of the exhaust gases, including synthesis gas, contain oxygen in small or large quantities. This reaction mixture makes it possible to obtain higher alcohols from the off-gas without having to go through the additional and expensive step of the initial oxygen evolution.

The reaction mixture according to any aspect of the present invention, thus, may contain CO, free oxygen and acetogenic bacteria in an exponential growth phase.

The person skilled in the art will understand the various phases of the growth of microorganisms and the methods for measuring and identifying them. In particular, most microorganisms in a batch culture can be in at least four different growth phases, namely the latent phase (A), the logarithmic phase or exponential phase (B), the stationary phase (C) and the death phase (D). The logarithmic phase can be further divided into the early logarithmic phase and the middle - late logarithmic / exponential phase. The stationary phase can also be further distinguished between the early stationary phase and the stationary phase. For example, Cotter J.L., 2009, Najafpour G., 2006, Younesi H., 2005, and Köpke M., 2009 reveal different phases of growth of acetogenic bacteria. In particular, the cell growth phase can be measured using the methods set out at least in Shuler M.L., 1992 and Fuchs G., 2007.

The latent phase is the phase immediately after inoculation of cells in a fresh environment, while the population remains temporarily unchanged. Although there is no visible cell division, cells can grow in volume or mass, synthesize enzymes, proteins, RNA, etc., and increase metabolic activity. The duration of the latent phase may depend on many different factors, including the size of the inoculum; the time required to recover from physical damage or shock during transfer; the time required for the synthesis of the most important coenzymes or division factors; and the time required for the synthesis of new (inducible) enzymes that are necessary for the metabolism of substrates present in the medium.

The exponential (logarithmic) phase of growth is a balanced growth model where all cells regularly divide by binary division and grow exponentially. Cells divide at a constant rate depending on the composition of the nutrient medium and incubation conditions. The rate of exponential growth of a bacterial culture is expressed as the time of generation, as well as the time of doubling of the bacterial population. Generation time (G) is defined as time (t) per generation (n = number of generations). Therefore, G = t / n is an equation by which the generation time calculations are obtained. The exponential phase can be divided into (i) the early logarithmic phase and (ii) the middle - late logarithmic / exponential phase. One skilled in the art can easily determine when a microorganism, in particular an acetogenic bacterium, enters the logarithmic phase. For example, a method for calculating the growth rate of acetogenic bacteria, determining whether they are in the logarithmic phase, can be performed using the method described at least in Henstra AM, 2007. In particular, a microorganism in an exponential growth phase according to any aspect of the present invention can include cells in the early logarithmic phase and in the middle - late logarithmic / exponential phase.

The stationary phase is the phase when exponential growth ends, because exponential growth cannot continue continuously in a batch culture (for example, in a closed system such as a test tube or flask). Population growth is limited by one of three factors: 1. exhaustion of available nutrients; 2. accumulation of inhibitory metabolites or end products; 3. exhaustion of space, in this case called the lack of "biological space". During the stationary phase, if viable cells are counted, it cannot be determined whether several cells die and an equal number of cells divide, or the population of cells simply stopped growing and dividing. The stationary phase, like the latent phase, is not necessarily a resting period. Bacteria that produce secondary metabolites, such as antibiotics, do this during the stationary phase of the growth cycle (secondary metabolites are defined as metabolites produced after the active growth stage).

The death phase follows the stationary phase. During the death phase, the number of viable cells decreases geometrically (exponentially), essentially back to growth during the logarithmic phase.

In one example, the acetogenic bacteria in the method according to any aspect of the present view may comprise a combination of cells: cells in a logarithmic phase and cells in a stationary phase. In the method according to any aspect of the present invention, the acetogenic cells in the logarithmic phase may contain a growth rate selected from the group consisting of 0.01 - 2 h ^-1 , 0.01 - 1 h ^-1 , 0.05 - 1 h ^-1 0.05 - 2 h ^-1 , 0.05 - 0.5 h ^-1 , etc. In one example, the OD ₆₀₀ cells of acetogenic cells in a logarithmic phase in the reaction mixture can be selected from the range consisting of 0.001 - 2, 0.01 - 2, 0.1 - 1, 0.1 - 0.5, etc. . One of skill in the art will be able to apply any method known in the art for measuring OD ₆₀₀ and determining the cell growth rate in the reaction mixture and / or to be added to the reaction mixture. For example, Koch (1994) can be used. In particular, bacterial growth can be determined and controlled using various methods. One of the most common is the measurement of turbidity, which relies on the optical density (OD) of bacteria in suspension and uses a spectrophotometer. OD can be measured at 600 nm using a UV spectrophotometer.

In one example, a method according to any aspect of the present invention comprises mixing (i) free oxygen, (ii) a group of acetogenic cells in a logarithmic phase, (iii) a group of acetogenic cells in a stationary phase, (iii) a group of Clostridium kluyveri and (iv) a carbon source containing CO, together. Acetogenic cells in the logarithmic phase allow any other acetogenic cells in the aqueous medium to produce acetate and / or ethanol in the presence of oxygen. The concentration of acetogenic cells in the logarithmic phase can be maintained in the reaction mixture. Thus, at any time in the reaction, the reaction mixture contains acetogenic cells in the logarithmic phase and acetogenic cells in another growth phase, for example, in the stationary phase.

The method according to any aspect of the present invention may further include the step of isolating the resulting higher alcohol. One skilled in the art will know the means to accomplish this based on methods known in the art.

According to another aspect of the present invention, there is provided the use of a reaction mixture according to any of the aspects of the present invention to produce at least one higher alcohol containing at least 6 carbon atoms. In particular, a higher alcohol is obtained from at least a carbon source containing CO.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

No figures

EXAMPLES

The foregoing describes preferred embodiments that, as will be appreciated by those skilled in the art, may be modified or modified in design, construction, and operation without departing from the scope of the claims. These changes, for example, are intended to be encompassed by the scope of the claims.

Example 1

Co-cultivation of Clostridium ljungdahlii and Clostridium kluyveri in a medium of a certain composition on hydrogen and carbon dioxide

In this example, C. ljungdahlii, as a first organism, was autotrophically cultured in a medium of a specific composition in order to produce acetate and ethanol. Then, after a predetermined time, C. kluyveri as a second organism was inoculated into the same reactor to convert acetate and ethanol to butyrate and hexanoate. Then C. ljungdahlii subsequently converted butyrate to butanol.

Used a medium of a certain composition for the joint cultivation of both microorganisms, consisting of 2 g / l (NH ₄ ) ₂ HPO ₄ , 0.2 g / l NaCl, 0.15 g / l KCl, 1 g / l KOH, 0.5 g / l MgCl ₂ x 6 H ₂ O, 0.2 g / l CaCl ₂ x 2 H ₂ O, 15 mg / l FeCl ₂ x 4 H ₂ O, 0.4 g / l L-cysteine-HCl, 0, 4 g / l Na ₂ S x 9 H ₂ O, 3 mg / l boric acid, 2 mg / l CoCl ₂ x 6 H ₂ O, 1 mg / l ZnSO ₄ x 7 H ₂ O, 0.3 mg / l Na ₂ MoO ₄ x 2 H ₂ O, 0.3 mg / L MnSO ₄ x H ₂ O, 0.2 mg / L NiCl ₂ x 6 H ₂ O, 0.1 mg / L CuCl ₂ x 2 H ₂ O , 0.1 mg / l Na ₂ SeO ₃ , 106 μg / l biotin, 5 μg / l folic acid, 2.5 μg / l pyridoxine-HCl, 266 μg / l thiamine-HCl x H ₂ O, 12.5 μg / L riboflavin, 12.5 μg / L nicotinic acid, 413 μg / L Ca-pantothenic acid, 12.5 μg / L Vitamin ₁₂ B, 12.5 mg / l p-aminobenzoic acid, 15 g / l lipoic acid.

Autotrophic cultivation was carried out in 250 ml of medium in 500 ml serum bottles, which were continuously aerated with synthesis gas consisting of 67% H ₂ and 33% CO ₂ at a rate of 1 l / h. Gas was introduced into the liquid phase using a microbubble disperser with a pore diameter of 10 μm. The whey bottle was continuously shaken in an Innova 3100 New Brunswick Scientific open water bath at 37 ° C. and a shaking speed of 150 min ⁻¹ . The pH value was maintained in the range of pH 5.0-6.5 by continuous addition of an anaerobic KOH stock solution (40 g / l).

At the beginning of the experiment, C. ljungdahlii was inoculated with OD _{600 of} 0.1 autotrophically grown cells. Thus, C. ljungdahlii was grown in a complex medium under continuous aeration with synthesis gas consisting of 67% H ₂ and 33% CO ₂ at a rate of 3 L / h in 1 liter serum bottles with 500 ml of complex medium. A complex medium was used consisting of 1 g / L NH ₄ Cl, 0.1 g / L KCl, 0.2 g / L MgSO ₄ x 7 H ₂ O, 0.8 g / L NaCl, 0.1 g / L KH ₂ PO ₄ , 20 mg / l CaCl ₂ x 2 H ₂ O, 20 g / l MES, 1 g / l yeast extract, 0.4 g / l L-cysteine-HCl, 0.4 g / l Na ₂ S x 9 H ₂ O, 20 mg / L nitrilotriacetic acid, 10 mg / L MnSO ₄ x H ₂ O, 8 mg / L (NH ₄ ) ₂ Fe (SO ₄ ) ₂ x 6 H ₂ O, 2 mg / L CoCl ₂ x 6 H ₂ O, 2 mg / L ZnSO ₄ x 7 H ₂ O, 0.2 mg / L CuCl ₂ x 2 H ₂ O, 0.2 mg / L Na ₂ MoO ₄ x 2 H ₂ O, 0.2 mg / L NiCl ₂ x 6 H ₂ O, 0.2 mg / L Na ₂ SeO ₄ , 0.2 mg / L Na ₂ WO ₄ x 2 H ₂ O, 20 μg / L Biotin, 20 μg / L of folic acid, 100 μg / L of pyridoxine-HCl, 50 μg / L of thiamine-HCl x H ₂ O, 50 μg / L of riboflavin, 50 μg / L of nicotinic acid, 50 μg / L of Ca-pantothenic acid, 1 μg / L vitamin on B ₁₂ , 50 μg / l p-aminobenzoic acid, 50 μg / l lipoic acid. Gas was introduced into the liquid phase using a microbubble disperser with a pore diameter of 10 μm. The whey bottle was continuously shaken in an Innova 3100 New Brunswick Scientific open water bath at 37 ° C. and a shaking speed of 150 min ⁻¹ . Cells were harvested in the late logarithmic phase with OD ₆₀₀ 0.67 and pH 4.69 using anaerobic centrifugation (4500 min ^-1 , 4300 g, 20 ° C, 10 min). The supernatant was removed, and the precipitate was again suspended in 10 ml of the above medium of a specific composition. This cell suspension was then used for inoculation in a co-culture experiment.

In parallel, C. kluyveri was grown heterotrophically in 200 ml of complex medium in 500 ml serum bottles on acetate and ethanol. A complex medium was used consisting of 0.25 g / L NH ₄ Cl, 0.2 g / L MgSO ₄ x 7 H ₂ O, 0.31 g / L K ₂ HPO ₄ , 0.23 g / L KH ₂ PO ₄ , 2.5 g / l NaHCO ₃ , 1 g / l yeast extract, 10 g / l K-acetate, 20 g / l ethanol, 0.25 g / l L-cysteine-HCl, 1.5 mg / l FeCl ₂ x 4 H ₂ O, 70 μg / L ZnCl ₂ x 7 H ₂ O, 100 μg / L MnCl ₂ x 4 H ₂ O, 6 μg / L Boric Acid, 190 μg / L CoCl ₂ x 6 H ₂ O , 2 μg / L CuCl ₂ x 6 H ₂ O, 24 μg / L NiCl ₂ x 6 H ₂ O, 36 μg / L Na ₂ MoO ₄ x 2 H ₂ O, 3 μg / L Na ₂ SeOO ₃ x 5 H2O , 4 μg / L Na ₂ WO ₄ x 2 H ₂ O, 100 μg / L Vitamin B ₁₂ , 80 μg / L p-aminobenzoic acid, 20 μg / L Biotin, 200 μg / L Nicotinic acid, 100 μg / L Ca -pantotenovoy acid, 300 mg / l pyridoxine-HCl, 200 mg / l thiamine-HCl x H ₂ O. The bottle for yvorotki continuously shaken in an open water bath Innova 3100 from New Brunswick Scientific at 37 ° C and shaking speed of 100 min ^-1. Cells were harvested in the late logarithmic phase with OD ₆₀₀ 0.81 and pH 5.96 using anaerobic centrifugation (4500 min ^-1 , 4300 g, 20 ° C, 10 min). The supernatant was removed, and the precipitate was again suspended in 10 ml of the above medium of a specific composition. This cell suspension was then used for inoculation in a co-culture experiment with an OD _{600 of} 0.2 after 96 hours of passage of the experiment.

During the experiment, 5 ml samples were taken to determine OD ₆₀₀ , pH, and product concentration. The latter was determined by quantitative analysis using ¹ H-NMR spectroscopy.

After inoculation of C. ljungdahlii, the cells began to grow and continuously produced acetate. Simultaneously with the production of acetate, ethanol was obtained at a lower rate compared to the production of acetate. Then, after 96 hours, C. kluyveri was inoculated into the reactor and the decrease in ethanol concentration was measured in the following experiment. Then, the production of butyrate (max. 1163 mg / l) and hexanoate (max. 136 mg / l) were simultaneously measured in the next 113 hours of the experiment. In parallel with the production of butyrate with C. kluyveri, C. ljungdahlii converted butyrate to butanol to a maximum concentration of 20 mg / l butanol at the end of the experiment.

Example 2

Co-cultivation of Clostridium ljungdahlii and Clostridium kluyveri in a complex medium with CO-containing gas (25% CO)

C. ljungdahlii as the first organism was autotrophically cultured in a complex medium for the production of acetate and ethanol. Then, after a predetermined time, C. kluyveri as a second organism was inoculated into the same reactor to convert acetate and ethanol to butyrate and hexanoate. Subsequently, C. ljungdahlii subsequently converted butyrate to butanol and hexanoate to hexanol.

Used a complex medium for the joint cultivation of both microorganisms, consisting of 1 g / l NH ₄ Cl, 0.1 g / l KCl, 0.2 g / l MgSO ₄ x 7 H ₂ O, 0.8 g / l NaCl, 0 , 1 g / l KH ₂ PO ₄ , 20 mg / l CaCl ₂ x 2 H ₂ O, 20 g / l MES, 1 g / l yeast extract, 0.4 g / l L-cysteine-HCl, 0.4 g / l Na ₂ S x 9 H ₂ O, 20 mg / l nitrilotriacetic acid, 10 mg / l MnSO ₄ x H ₂ O, 8 mg / l (NH ₄ ) ₂ Fe (SO ₄ ) ₂ x 6 H ₂ O , 2 mg / L CoCl ₂ x 6 H ₂ O, 2 mg / L ZnSO ₄ x 7 H ₂ O, 0.2 mg / L CuCl ₂ x 2 H ₂ O, 0.2 mg / L Na ₂ MoO ₄ x 2 H ₂ O, 0.2 mg / L NiCl ₂ x 6 H ₂ O, 0.2 mg / L Na ₂ SeO ₄ , 0.2 mg / L Na ₂ WO ₄ x 2 H ₂ O, 20 μg / L biotin, 20 g / l of folic acid, 100 micrograms / l pyridoxine-HCl, 50 mg / l thiamine-HCl x H ₂ O, 50 g / l of riboflavin, 50 g / l nicotine oh acid 50 g / L Ca-pantothenic acid, 1 mg / l of vitamin B _12, 50 mcg / l p-aminobenzoic acid, 50 g / l lipoic acid.

Autotrophic cultivation was performed in 500 ml of complex medium in a 1 L serum bottle, which was continuously gassed with synthesis gas consisting of 5% H ₂ , 25% CO ₂ , 25% CO and 45% N ₂ , at a rate of ~ 3.6 l / h (≥0.5 ppm gaseous oxygen). Gas was introduced into the liquid phase using a microbubble disperser with a pore diameter of 10 μm. The serum bottle was continuously shaken in an Innova 3100 New Brunswick Scientific open water bath at 37 ° C. and a shaking speed of 120 min ⁻¹ . The pH value was not controlled during this experiment.

At the beginning of the experiment, C. ljungdahlii was inoculated with OD _{600 of} 0.1 autotrophically grown cells. Thus, C. ljungdahlii was grown in the complex medium described above under continuous aeration with synthesis gas consisting of 67% H ₂ and 33% CO ₂ at a rate of 3 l / h in 1 liter serum bottles with 500 ml of complex medium. Gas was introduced into the liquid phase using a microbubble disperser with a pore diameter of 10 μm. The whey bottle was continuously shaken in an Innova 3100 New Brunswick Scientific open water bath at 37 ° C. and a shaking speed of 150 min ⁻¹ . Cells were harvested in the late logarithmic phase with OD ₆₀₀ 0.51 and pH 5.04 by anaerobic centrifugation (4500 min ^-1 , 4300 g, 20 ° C, 10 min). The supernatant was removed and the precipitate was again suspended in 10 ml of the above complex medium. This cell suspension was then used for inoculation in a co-culture experiment.

In parallel, C. kluyveri was grown heterotrophically in 200 ml of complex medium in 500 ml serum bottles on acetate and ethanol. A complex medium was used consisting of 0.25 g / L NH ₄ Cl, 0.2 g / L MgSO ₄ x 7 H ₂ O, 0.31 g / L K ₂ HPO ₄ , 0.23 g / L KH ₂ PO ₄ , 2.5 g / l NaHCO ₃ , 1 g / l yeast extract, 10 g / l K-acetate, 20 g / l ethanol, 0.25 g / l L-cysteine-HCl, 1.5 mg / l FeCl ₂ x 4 H ₂ O, 70 μg / L ZnCl ₂ x 7 H ₂ O, 100 μg / L MnCl ₂ x 4 H ₂ O, 6 μg / L Boric Acid, 190 μg / L CoCl ₂ x 6 H ₂ O , 2 μg / L CuCl ₂ x 6 H ₂ O, 24 μg / L NiCl ₂ x 6 H ₂ O, 36 μg / L Na ₂ MoO ₄ x 2 H ₂ O, 3 μg / L Na ₂ MoO ₄ x 5 H ₂ O, 4 μg / L Na ₂ WO ₄ x 2 H ₂ O, 100 μg / L Vitamin B ₁₂ , 80 μg / L p-aminobenzoic acid, 20 μg / L Biotin, 200 μg / L Nicotinic acid, 100 μg / l Ca-pantothenic acid, 300 μg / l pyridoxine-HCl, 200 μg / l thiamine-HCl x H ₂ O. Bottle for the sera were continuously shaken in an Innova 3100 New Brunswick Scientific open water bath at 37 ° C. and a shaking speed of 100 min ⁻¹ . Cells were harvested in the late logarithmic phase with an OD _{600 of} 0.54 and a pH of 6.60 by anaerobic centrifugation (4500 min ^-1 , 4300 g, 20 ° C, 10 min). The supernatant was removed and the precipitate was again suspended in 10 ml of the above complex medium. This cell suspension was then used for inoculation in a co-culture experiment after 240 hours of experiment passage.

During the experiment, 5 ml samples were taken to determine OD ₆₀₀ , pH, and product concentration. The latter was determined by quantitative analysis using ¹ H-NMR spectroscopy.

After inoculation of C. ljungdahlii, cells began to grow and continuously produced acetate to a concentration of ~ 3 g / L and ethanol to a concentration of ~ 0.5 g / L after 71 hours. In the further dynamics of the experiment, acetate was completely converted to ethanol to a concentration of 4.8 g / l after 240 hours. Then, at the time of the process, 240 hours C. kluyveri was inoculated into the reactor. Since this organism, in addition to ethanol, needed acetate as a substrate, simultaneously with the inoculation of C. kluyveri, approximately 3 g / l of acetate (in the form of Na-acetate) was anaerobically introduced into the reactor. In the further dynamics of the experiment, the production of butyrate and hexanoate was measured to a concentration of each of 1.6 g / L. In parallel with the production of butyrate and hexanoate with C. kluyveri, C. ljungdahlii converted butyrate to butanol to a maximum concentration of 690 mg / L butanol and converted hexanoate to hexanol to a maximum concentration of 1478 mg / L hexanol.

Example 3

Obtaining acetate and ethanol using Clostridium ljungdahlii from synthesis gas without oxygen

In this example, C. ljungdahlii was anaerobically cultured in a complex medium with synthesis gas consisting of H ₂ and CO ₂ in the absence of oxygen in order to obtain acetate and ethanol. For C. ljungdahlii cell culture, 2 ml of cryoculture was cultured anaerobically in 200 ml of medium (ATCC1754 medium: pH 6.0; 20 g / l MES; 1 g / l yeast extract, 0.8 g / l NaCl, 1 g / l NH ₄ Cl, 0.1 g / L KCl, 0.1 g / L KH ₂ PO ₄ , 0.2 g / L MgSO ₄ x 7 H ₂ O; 0.02 g / L CaCl ₂ × 2H ₂ O; 20 mg / l nitrilotriacetic acid, 10 mg / l MnSO ₄ x H ₂ O; 8 mg / l (NH ₄ ) ₂ Fe (SO ₄ ) ₂ x 6 H ₂ O; 2 mg / l CoCl ₂ x 6 H ₂ O; 2 mg / L ZnSO ₄ x 7 H ₂ O; 0.2 mg / L CuCl ₂ x 2 H ₂ O; 0.2 mg / L Na ₂ MoO ₄ x 2 H ₂ O; 0.2 mg / L NiCl ₂ x 6 H ₂ O; 0.2 mg / L Na ₂ SeO ₄ ; 0.2 mg / L Na ₂ WO ₄ x 2 H ₂ O; 20 μg / L d-Biotin, 20 μg / L Folic Acid, 100 g / l pyridoxine-HCl; 50 μg / l thiamine-HCl x H ₂ O; 50 μg / l riboflavin; 50 μg / l nicotinic acid, 50 μg / l Ca-pantothenate; 1 μg / L Vitamin B12; 50 μg / L p-aminobenzoate; 50 μg / L Lipoic acid, approximately 67.5 mg / L NaOH) with approximately 400 mg / L L-cysteine hydrochloride and 400 mg / L Na ₂ S x 9 H ₂ O. The cultivation was carried out chemolithoautotrophically in a 1 L fire-resistant glass bottle with a pre-mixed gas mixture consisting of 67% H ₂ , 33% CO ₂ in a shaker in an open water bath at 37 ° C, 150 rpm. and fumigation of 1-3 l / h for 161 hours. The introduction of gas into the medium was carried out using a filter with a pore size of 10 microns, installed in the middle of the reactor in a gas discharge pipe. Cells were centrifuged, washed with 10 ml of ATCC medium and centrifuged again.

For preculture, many washed cells from a grown C. ljungdahlii culture were transferred to 200 ml of ATCC medium with approximately 400 mg / L L-cysteine hydrochloride and grown to OD ₆₀₀ 0.12. The cultivation was carried out in a pressure-resistant 500 ml glass bottle with a pre-mixed gas mixture consisting of 67% H ₂ , 33% CO ₂ in a shaker in an open water bath at 37 ° C, 150 rpm. and aeration of 3 l / h for 65 hours. The gas was introduced into the medium using a filter with a pore size of 10 microns, which was placed in the middle of the reactors. The cells were centrifuged, washed with 10 ml of production buffer (pH 6.2; 0.5 g / l KOH, aerated for 1 h with a pre-mixed mixture of gases from 67% H ₂ , 33% CO ₂ at 1 l / h), again washed and centrifuged.

For a producing culture, many washed cells from the C. ljungdahlii preculture were transferred to 200 ml of ATCC medium with approximately 400 mg / L L-cysteine hydrochloride and grown to OD ₆₀₀ 0.2. The cultivation was carried out in a pressure-resistant 500 ml glass bottle with a pre-mixed gas mixture consisting of 67% H ₂ , 33% CO ₂ in a shaker in an open water bath at 37 ° C, 150 rpm. and aeration of 3 l / h for 118 hours. The gas was introduced into the medium using a filter with a pore size of 10 microns, which was placed in the middle of the reactors. When the pH dropped below 5.0, 1 ml of a solution of 140 g / l KOH was added. When taking samples, each 5 ml sample was removed to determine OD _600, pH, and product range. Determination of the concentration of the product was carried out using semi-quantitative analysis using 1H-NMR spectroscopy. Sodium trimethylsilyl propionate (T (M) SP) was used as an internal quantitative standard.

During the cultivation period of 118 h, the cell density in the producing culture remained constant, recognizable by a standard OD _{600 of} 0.2, which corresponds to a growth rate of μ = 0 h ^-1 . The concentration of acetate increased significantly over this period of time from 4 mg / L to 3194 mg / L and the concentration of ethanol increased from 17 mg / L to 108 mg / L.

Example 4

Lack of acetate and ethanol production with Clostridium ljungdahlii from synthesis gas containing CO ₂ and H ₂ with oxygen

C. ljungdahlii was cultured in a complex medium with synthesis gas and oxygen. C. ljungdahlii was initially cultured in the presence of synthesis gas consisting of H ₂ and CO ₂ in the absence of oxygen to produce acetate and ethanol. For cultivation, cells were grown in pressure-resistant glass bottles, which can be sealed with a butyl rubber stopper. All stages in which C. ljungdahlii cells were used were performed under anaerobic conditions.

For C. ljungdahlii cell culture, 2 ml of cryoculture was cultured anaerobically in 200 ml of medium (ATCC1754 medium: pH 6.0; 20 g / l MES; 1 g / l yeast extract, 0.8 g / l NaCl, 1 g / l NH ₄ Cl, 0.1 g / L KCl, 0.1 g / L KH ₂ PO ₄ , 0.2 g / L MgSO ₄ x 7 H ₂ O; 0.02 g / L CaCl ₂ × 2H ₂ O; 20 mg / l nitrilotriacetic acid, 10 mg / l MnSO ₄ x H ₂ O; 8 mg / l (NH ₄ ) ₂ Fe (SO ₄ ) ₂ x 6 H ₂ O; 2 mg / l CoCl ₂ x 6 H ₂ O; 2 mg / L ZnSO ₄ x 7 H ₂ O; 0.2 mg / L CuCl ₂ x 2 H ₂ O; 0.2 mg / L Na ₂ MoO ₄ x 2 H ₂ O; 0.2 mg / L NiCl ₂ x 6 H ₂ O; 0.2 mg / L Na ₂ SeO ₄ ; 0.2 mg / L Na ₂ WO ₄ x 2 H ₂ O; 20 μg / L d-Biotin, 20 μg / L Folic Acid, 100 g / l pyridoxine-HCl; 50 μg / l thiamine-HCl x H ₂ O; 50 μg / l riboflavin; 50 μg / l nicotinic acid, 50 μg / l Ca-pantothenate; 1 μg / L Vitamin B12; 50 μg / L p-aminobenzoate; 50 μg / L Lipoic acid, approximately 67.5 mg / L NaOH) with approximately 400 mg / L L-cysteine hydrochloride and 400 mg / L Na ₂ S x 9 H ₂ O. The cultivation was carried out chemolithoautotrophically in a 1 L fire-resistant glass bottle with a pre-mixed gas mixture consisting of 67% H ₂ , 33% CO ₂ in a shaker in an open water bath at 37 ° C, 150 rpm. and fumigation of 1-3 l / h for 161 hours. The introduction of gas into the medium was carried out using a filter with a pore size of 10 microns, installed in the middle of the reactor in a gas discharge pipe. Cells were centrifuged, washed with 10 ml of ATCC medium and centrifuged again.

For preculture, many washed cells from a grown C. ljungdahlii culture were transferred to 200 ml of ATCC medium with approximately 400 mg / L L-cysteine hydrochloride and grown to OD ₆₀₀ 0.12. The cultivation was carried out in a pressure-resistant 500 ml glass bottle with a pre-mixed gas mixture consisting of 67% H ₂ , 33% CO ₂ in a shaker in an open water bath at 37 ° C, 150 rpm. and with aeration of 3 l / h for 24 hours. Subsequently, the gas mixture was replaced by a mixture with a composition of 66.85% H ₂ , 33% CO ₂ and 0.15% O ₂ and the cells were additionally aerated for 67 h at 3 l / h The gas was introduced into the medium using a carbonating frit with a pore size of 10 microns, which was placed in the middle of the reactors on a bubbler. Cells were centrifuged, washed with 10 ml of ATCC medium and centrifuged again. The gas was introduced into the medium using a filter with a pore size of 10 microns, which was placed in the middle of the reactors. Cells were centrifuged, washed with 10 ml of ATCC medium and centrifuged again.

For a producing culture, many washed cells from the C. ljungdahlii preculture were transferred to 200 ml of ATCC medium with approximately 400 mg / L L-cysteine hydrochloride and grown to OD ₆₀₀ 0.1. The cultivation was carried out in a pressure-resistant 500 ml glass bottle with a pre-mixed mixture of gases consisting of 66.85% H ₂ , 33% CO ₂ and 0.15% O ₂ , in a shaker in an open water bath at 37 ° C, 150 rpm and with aeration of 3 l / h for 113 hours. The gas was introduced into the medium using a filter with a pore size of 10 microns, which was placed in the middle of the reactors. When taking samples, each 5 ml sample was removed to determine OD _600, pH, and product range. Determination of the concentration of the product was carried out using semi-quantitative analysis using 1H-NMR spectroscopy. Sodium trimethylsilyl propionate (T (M) SP) was used as an internal quantitative standard.

For the period from 89 h to 113 h, no recognizable cell growth was shown. OD ₆₀₀ stopped at 0.29, which corresponds to a growth rate of μ = 0 h ^-1 . The acetate concentration slightly increased during this time from 89.4 mg / L to 86.9 mg / L and the ethanol concentration decreased from 16.2 mg / L to 11.9 mg / L.

Example 5

Cultivation of Clostridium ljungdahlii in the logarithmic phase in the presence of synthesis gas containing CO ₂ and oxygen

C. ljungdahlii fed on H ₂ and CO ₂ from the penetrating gas phase and formed acetate and ethanol. For cultivation, a pressure-resistant glass bottle was used, which can be sealed using a butyl rubber stopper. All cultivation steps in which C. ljungdahlii cells were used were performed under anaerobic conditions.

For C. ljungdahlii cell culture, 5 ml of cryoculture was cultured anaerobically in 500 ml of medium (ATCC1754 medium: pH 6.0; 20 g / l MES; 1 g / l yeast extract, 0.8 g / l NaCl, 1 g / l NH ₄ Cl, 0.1 g / L KCl, 0.1 g / L KH ₂ PO4, 0.2 g / L MgSO ₄ x 7 H ₂ O; 0.02 g / L CaCl ₂ × 2H ₂ O; 20 mg / l nitrilotriacetic acid, 10 mg / l MnSO ₄ x H ₂ O; 8 mg / l (NH ₄ ) 2Fe (SO ₄ ) ₂ x 6 H ₂ O; 2 mg / l CoCl ₂ x 6 H ₂ O; 2 mg / l ZnSO ₄ x 7 H ₂ O; 0.2 mg / l CuCl ₂ x 2 H ₂ O; 0.2 mg / l Na ₂ MoO ₄ x 2 H ₂ O; 0.2 mg / l NiCl ₂ x 6 H ₂ O; 0.2 mg / L Na ₂ SeO ₄ ; 0.2 mg / L Na ₂ WO ₄ x 2 H ₂ O; 20 μg / L d-Biotin, 20 μg / L Folic Acid, 100 g / L pyridoxine-HCl; 50 μg / L thiamine-HCl x H2O; 50 μg / L riboflavin; 50 μg / L nicotinic acid, 50 μg / L Ca pantothenate; 1 μg / L vitamin B12; 50 μg / L p-aminobenzoate; 50 μg / L lipoic acid, approximately 67.5 mg / L NaOH) with approximately 400 mg / L L-cysteine hydrochloride and 400 mg / L Na ₂ S x 9 H ₂ O. The cultivation was carried out chemolithoautotrophically in a 1 liter fire-resistant glass bottle with a pre-mixed gas mixture consisting of 67% H ₂ , 33% CO ₂ in a shaker in an open water bath at 37 ° C, 100 rpm. and fumigation of 3 l / h for 72 hours. The gas was introduced into the medium using a filter with a pore size of 10 microns installed in the middle of the reactor in a gas discharge pipe. Cells were centrifuged, washed with 10 ml of ATCC medium and centrifuged again.

For the main culture, many washed cells from the grown C. ljungdahlii culture were transferred to 500 ml ATCC medium with approximately 400 mg / L L-cysteine hydrochloride and grown to OD ₆₀₀ 0.1. The cultivation was carried out in a pressure-resistant 1 liter glass bottle with a pre-mixed gas mixture consisting of 66.85% H ₂ , 33% CO ₂ , 0.15% O ₂ , in a shaker in an open water bath at 37 ° C, 150 rpm and with aeration of 1 l / h for 45 hours. The gas was introduced into the medium using a filter with a pore size of 10 microns, which was placed in the middle of the reactors. When taking samples, each 5 ml sample was removed to determine OD _600nm , pH and product range. Determination of the concentration of the product was carried out using semi-quantitative analysis using 1H-NMR spectroscopy. Sodium trimethylsilyl propionate (T (M) SP) was used as an internal quantitative standard.

Significant cell growth was shown during the cultivation period, as evidenced by an increase in OD _600nm from 0.10 to 0.54, which corresponded to a growth rate of μ = 0.037 h ^-1 . The acetate concentration increased over this period of time from 9.6 mg / L to 3304 mg / L and the concentration of ethanol increased from 2.2 mg / L to 399 mg / L.

Example 6

Cultivation of Clostridium ljungdahlii in the logarithmic phase in the presence of synthesis gas containing CO and oxygen (65% CO)

C. ljungdahlii was autotrophically cultured in a complex medium with a synthesis gas consisting of CO, H ₂ and CO ₂ , in the presence of oxygen, to produce acetate and ethanol.

A complex medium was used consisting of 1 g / L NH ₄ Cl, 0.1 g / L KCl, 0.2 g / L MgSO ₄ x 7 H ₂ O, 0.8 g / L NaCl, 0.1 g / L KH ₂ PO ₄ , 20 mg / l CaCl ₂ x 2 H ₂ O, 20 g / l MES, 1 g / l yeast extract, 0.4 g / l L-cysteine-HCl, 0.4 g / l Na ₂ S x 9 H ₂ O, 20 mg / L nitrilotriacetic acid, 10 mg / L MnSO ₄ x H ₂ O, 8 mg / L (NH ₄ ) ₂ Fe (SO ₄ ) ₂ x 6 H ₂ O, 2 mg / L CoCl ₂ x 6 H ₂ O, 2 mg / L ZnSO ₄ x 7 H ₂ O, 0.2 mg / L CuCl ₂ x 2 H ₂ O, 0.2 mg / L Na ₂ MoO ₄ x 2 H ₂ O, 0.2 mg / L NiCl ₂ x 6 H ₂ O, 0.2 mg / L Na ₂ SeO ₄ , 0.2 mg / L Na ₂ WO ₄ x 2 H ₂ O, 20 μg / L Biotin, 20 μg / L of folic acid, 100 μg / L of pyridoxine-HCl, 50 μg / L of thiamine-HCl x H ₂ O, 50 μg / L of riboflavin, 50 μg / L of nicotinic acid, 50 μg / L of Ca-pantothenic acid, 1 μg / L vitamin on B ₁₂ , 50 μg / l p-aminobenzoic acid, 50 μg / l lipoic acid.

Autotrophic cultivation was carried out in 500 ml of medium in 1 L serum bottles, which were continuously gassed with synthesis gas consisting of 65% CO, 4% H ₂ and 15% CO ₂ at a rate of 3.6 l / h. Gas was introduced into the liquid phase using a microbubble disperser with a pore diameter of 10 μm. The serum bottle was continuously shaken in an Innova 3100 New Brunswick Scientific open water bath at 37 ° C. and a shaking speed of 120 min ⁻¹ . The pH value was not controlled.

At the beginning of the experiment, C. ljungdahlii was inoculated with OD ₆₀₀ 0.1 autotrophically grown on H ₂ / CO ₂ cells. Thus, C. ljungdahlii was grown in a complex medium under continuous aeration with synthesis gas consisting of 67% H ₂ and 33% CO ₂ at a rate of 3 L / h in 1 liter serum bottles with 500 ml of complex medium. The above medium was also used for this cultivation. Gas was introduced into the liquid phase using a microbubble disperser with a pore diameter of 10 μm. The whey bottle was continuously shaken in an Innova 3100 New Brunswick Scientific open water bath at 37 ° C. and a shaking speed of 150 min ⁻¹ . Cells were harvested in a logarithmic phase with OD ₆₀₀ 0.49 and pH 5.03 by anaerobic centrifugation (4500 min ^-1 , 4300 g, 20 ° C, 10 min). The supernatant was removed and the precipitate was again suspended in 10 ml of the above medium. This cell suspension was then used for inoculation in a culture experiment. Carbon monoxide gas phase concentration was measured by gas phase sampling and autonomous analysis using a GC 6890N gas chromatograph from Agilent Technologies Inc. with a thermal conductivity detector The concentration of the oxygen gas phase was measured using an immersion probe for trace amounts of oxygen from PreSens Precision Sensing GmbH. The oxygen concentration was measured using quenching of fluorescence, where the degree of quenching is correlated with the partial pressure of oxygen in the gas phase. Oxygen measurements indicated a concentration of 0.1 vol.% O ₂ in the synthesis gas used.

During the experiment, 5 ml samples were taken to determine OD ₆₀₀ , pH, and product concentration. The latter was determined by quantitative analysis using ¹ H-NMR spectroscopy.

After inoculation of C. ljungdahlii, cells began to grow at a growth rate of μ = 0.062 h ^-1 and acetate was continuously produced to a concentration of 6.2 g / l after 94.5 hours. Simultaneously with the production of acetate, ethanol was obtained at a lower rate compared to the production of acetate to a concentration of 1 g / l after 94.5 hours.

  Application of NMR analysis Process time, h pH OD600 Acetate,
mg / l Ethanol
mg / l 0,0 6.15 0.10 18 but. 18.0 5.97 0.69 973 97 42.5 5.20 1,50 66.0 4.67 1.95 5368 966 94.5 4,54 1.77 6187 1070

Table 3. The results of Example 6 (n.o. = not detected)

Example 7

Growth and acetate production in Clostridium ljungdahlii on synthesis gas with oxygen

For biotransformation of hydrogen and carbon dioxide into acetic acid, the homoacetogenic bacterium Clostridium ljungdahlii was cultivated on synthesis gas with oxygen. All stages of cultivation were carried out under anaerobic conditions in pressure-resistant glass bottles, which can be hermetically sealed with a butyl rubber stopper.

For preculture in 500 ml of medium (ATCC1754-medium: pH = 6.0; 20 g / l MES; 1 g / l yeast extract, 0.8 g / l NaCl; 1 g / l NH ₄ Cl; 0.1 g / l KCl; 0.1 g / l KH ₂ PO4; 0.2 g / l MgSO ₄ x 7 H ₂ O; 0.02 g / l CaCl ₂ x 2 H ₂ O; 20 mg / l nitrilotriacetic acid, 10 mg / L MnSO ₄ x H ₂ O; 8 mg / L (NH ₄ ) ₂ Fe (SO ₄ ) ₂ x 6 H ₂ O; 2 mg / L CoCl ₂ x 6 H ₂ O; 2 mg / L ZnSO ₄ x 7 H ₂ O; 0.2 mg / L CuCl ₂ x 2 H ₂ O; 0.2 mg / L Na ₂ MoO ₄ x 2 H ₂ O; 0.2 mg / L NiCl ₂ x 6 H ₂ O; 0 , 2 mg / L Na ₂ SeO ₄ ; 0.2 mg / L Na ₂ WO ₄ x 2 H ₂ O; 20 μg / L d-Biotin, 20 μg / L Folic Acid, 100 μg / L Pyridoxine-HCl; 50 μg / L thiamine-HCl x H ₂ O; 50 μg / L riboflavin; 50 μg / L nicotinic acid, 50 μg / L Ca-pantothenate; 1 μg / L Vitamin B ₁₂ ; 50 μg / L p-aminobenzoate; 50 μg / l lip Eve acid, about 67.5 mg / l NaOH) with additional 400 mg / l L-cysteine hydrochloride and 400 mg / l Na ₂ S x 9 H ₂ O was inoculated with 5 ml of frozen raw krioshtamma C. ljungdahlii. Chemolithoautotrophic cultivation was carried out in a pressure-resistant 1 liter glass bottle at 37 ° C, 100 rpm. and a ventilation rate of 3 l / h with pre-mixed gas with 67% H ₂ , 33% CO ₂ in a shaker in an open water bath for 72 hours. The gas was released into the medium through a bubbler with a pore size of 10 μm, which was installed in the central part of the reactors . Cultivation was carried out without pH control.

After preliminary cultivation, the cell suspension was centrifuged (10 min, 4200 rpm) and the pellet was washed with 10 ml of medium and centrifuged again. For the main culture, the required number of washed cells from the preculture, for OD _{600 nm} 0.1, was transferred to 200 ml of medium with an additional 400 mg / L of L-cysteine hydrochloride. Chemolithoautotrophic cultivation was carried out in pressure-resistant glass bottles with a volume of 250 ml at 37 ° C, 150 rpm. and a ventilation rate of 1 l / h with pre-mixed gas with 65% H ₂ , 33% CO ₂ , 2% O ₂ in a shaker in an open water bath for 47 hours. The gas was released into the medium through a bubbler with a pore size of 10 μm, which was installed in the central part of the reactors. Cultivation was carried out without pH control. During cultivation, several 5 ml samples were taken to determine OD _600nm , pH and product formation. Determination of the concentration of the products was carried out using semi-quantitative analysis using 1H-NMR spectroscopy. Sodium trimethylsilyl propionate (T (M) SP) was used as an internal quantitative standard. Dissolved oxygen in the cultivation medium was also measured in real time using oxygen immersion probes (PSt6 with Oxy4Trace, Presens, Germany).

During the cultivation period, cell growth was observed by increasing OD _{600 nm} from 0.11 to 0.32, which correlated with a growth rate of µ = 0.022 h ^-1 . The concentration of acetate increased from 8 mg / L to 91 mg / L; no increase in ethanol concentration was observed. During the cultivation period, the concentration of dissolved oxygen ranged from 0.06 to 0.15 mg / L.

In a similar technical setting with the same parameters (medium composition, volume, bottle, gas, ventilation rate, temperature, frequency of shaking), but without cells in the medium, the concentration of dissolved oxygen 0.50 mg / L was measured.

Example 8

The growth and production of acetate in Clostridium ljungdahlii on synthesis gas with oxygen

For biotransformation of hydrogen and carbon dioxide into acetic acid, the homoacetogenic bacterium Clostridium ljungdahlii was cultivated on synthesis gas with oxygen. All stages of cultivation were carried out under anaerobic conditions in pressure-resistant glass bottles, which can be hermetically sealed with a butyl rubber stopper.

For preculture in 500 ml of medium (ATCC1754-medium: pH = 6.0; 20 g / l MES; 1 g / l yeast extract, 0.8 g / l NaCl; 1 g / l NH ₄ Cl; 0.1 g / l KCl; 0.1 g / l KH ₂ PO4; 0.2 g / l MgSO ₄ x 7 H ₂ O; 0.02 g / l CaCl ₂ x 2 H ₂ O; 20 mg / l nitrilotriacetic acid, 10 mg / L MnSO ₄ x H ₂ O; 8 mg / L (NH ₄ ) ₂ Fe (SO ₄ ) ₂ x 6 H ₂ O; 2 mg / L CoCl ₂ x 6 H ₂ O; 2 mg / L ZnSO ₄ x 7 H ₂ O; 0.2 mg / L CuCl ₂ x 2 H ₂ O; 0.2 mg / L Na ₂ MoO ₄ x 2 H ₂ O; 0.2 mg / L NiCl ₂ x 6 H ₂ O; 0 , 2 mg / L Na ₂ SeO ₄ ; 0.2 mg / L Na ₂ WO ₄ x 2 H ₂ O; 20 μg / L d-Biotin, 20 μg / L Folic Acid, 100 μg / L Pyridoxine-HCl; 50 μg / L thiamine-HCl x H ₂ O; 50 μg / L riboflavin; 50 μg / L nicotinic acid, 50 μg / L Ca-pantothenate; 1 μg / L Vitamin B ₁₂ ; 50 μg / L p-aminobenzoate; 50 μg / l lip Eve acid, about 67.5 mg / l NaOH) with additional 400 mg / l L-cysteine hydrochloride and 400 mg / l Na ₂ S x 9 H ₂ O was inoculated with 5 ml of frozen raw krioshtamma C. ljungdahlii. Chemolithoautotrophic cultivation was carried out in a pressure-resistant 1 liter glass bottle at 37 ° C, 100 rpm. and a ventilation rate of 3 l / h with pre-mixed gas with 67% H ₂ , 33% CO ₂ in a shaker in an open water bath for 72 hours. The gas was released into the medium through a bubbler with a pore size of 10 μm, which was installed in the central part of the reactors . Cultivation was carried out without pH control.

After preliminary cultivation, the cell suspension was centrifuged (10 min, 4200 rpm) and the pellet was washed with 10 ml of medium and centrifuged again. For the main culture, the required number of washed cells from the preculture, for OD _{600 nm} 0.1, was transferred to 200 ml of medium with an additional 400 mg / L of L-cysteine hydrochloride. Chemolithoautotrophic cultivation was carried out in pressure-resistant glass bottles with a volume of 250 ml at 37 ° C, 150 rpm. and a ventilation rate of 1 l / h with pre-mixed gas with 66.85% H ₂ , 33% CO ₂ , 0.15% O ₂ in a shaker in an open water bath for 47 hours. The gas was released into the medium through a bubbler with a pore size of 10 microns, which was installed in the central part of the reactors. Cultivation was carried out without pH control. During cultivation, several 5 ml samples were taken to determine OD _{600 nm} , pH, and product formation. Determination of the concentration of the products was carried out using semi-quantitative analysis using 1H-NMR spectroscopy. Sodium trimethylsilyl propionate (T (M) SP) was used as an internal quantitative standard. Dissolved oxygen in the cultivation medium was also measured in real time using oxygen immersion probes (PSt6 with Oxy4Trace, Presens, Germany).

During the cultivation period, cell growth was observed by increasing the OD _{600 nm} from 0.10 to 0.45, which correlated with a growth rate of µ = 0.032 h ^-1 . The acetate concentration increased from 7 mg / L to 2347 mg / L and the ethanol concentration increased from 2 mg / L to 319 mg / L. Over the entire cultivation period, the concentration of dissolved oxygen was 0.00 mg / L.

In a similar technical setting with the same parameters (medium composition, volume, bottle, gas, ventilation rate, temperature, frequency of shaking), but without cells in the medium, the concentration of dissolved oxygen of 0.03 mg / L was measured.

Example 9

Co-cultivation of Clostridium ljungdahlii and Clostridium kluyveri in a complex medium with CO-containing gas (7% CO)

C. ljungdahlii as the first organism was autotrophically cultured in a complex medium to produce acetate and ethanol. Then, after a predetermined time, C. kluyveri as a second organism was inoculated into the same reactor to convert acetate and ethanol to butyrate and hexanoate. Subsequently, C. ljungdahlii subsequently converted butyrate to butanol and hexanoate to hexanol.

Used a complex medium for the joint cultivation of both microorganisms, consisting of 1 g / l NH ₄ Cl, 0.1 g / l KCl, 0.2 g / l MgSO ₄ x 7 H ₂ O, 0.8 g / l NaCl, 0 , 1 g / l KH ₂ PO ₄ , 20 mg / l CaCl ₂ x 2 H ₂ O, 20 g / l MES, 1 g / l yeast extract, 0.4 g / l L-cysteine-HCl, 0.4 g / l Na ₂ S x 9 H ₂ O, 20 mg / l nitrilotriacetic acid, 10 mg / l MnSO ₄ x H ₂ O, 8 mg / l (NH ₄ ) ₂ Fe (SO ₄ ) ₂ x 6 H ₂ O , 2 mg / L CoCl ₂ x 6 H ₂ O, 2 mg / L ZnSO ₄ x 7 H ₂ O, 0.2 mg / L CuCl ₂ x 2 H ₂ O, 0.2 mg / L Na ₂ MoO ₄ x 2 H ₂ O, 0.2 mg / L NiCl ₂ x 6 H ₂ O, 0.2 mg / L Na ₂ SeO ₄ , 0.2 mg / L Na ₂ WO ₄ x 2 H ₂ O, 20 μg / L biotin, 20 g / l of folic acid, 100 micrograms / l pyridoxine-HCl, 50 mg / l thiamine-HCl x H ₂ O, 50 g / l of riboflavin, 50 g / l nicotine oh acid 50 g / L Ca-pantothenic acid, 1 mg / l of vitamin B _12, 50 mcg / l p-aminobenzoic acid, 50 g / l lipoic acid.

Autotrophic cultivation was performed in 500 ml of complex medium in a 1 L serum bottle, which was continuously gassed with synthesis gas consisting of 63% H ₂ , 7% CO ₂ and 2% CO, at a rate of ~ 3.6 l / h (≥ 0.5 ppm oxygen). Gas was introduced into the liquid phase using a microbubble disperser with a pore diameter of 10 μm. The serum bottle was continuously shaken in an Innova 3100 New Brunswick Scientific open water bath at 37 ° C. and a shaking speed of 120 min ⁻¹ . The pH value was not controlled during this experiment.

At the beginning of the experiment, C. ljungdahlii was inoculated with OD _{600 of} 0.1 autotrophically grown cells. Thus, C. ljungdahlii was grown in the complex medium described above under continuous aeration with synthesis gas consisting of 67% H ₂ and 33% CO ₂ at a rate of 3 l / h in 1 liter serum bottles with 500 ml of complex medium. Gas was introduced into the liquid phase using a microbubble disperser with a pore diameter of 10 μm. The whey bottle was continuously shaken in an Innova 3100 New Brunswick Scientific open water bath at 37 ° C. and a shaking speed of 150 min ⁻¹ . Cells were harvested in a stationary phase with OD ₆₀₀ 0.89 and pH 4.52 using anaerobic centrifugation (4500 min ^-1 , 4300 g, 20 ° C, 10 min). The supernatant was removed and the precipitate was again suspended in 10 ml of the above complex medium. This cell suspension was then used for inoculation in a co-culture experiment.

In parallel, C. kluyveri was grown heterotrophically in 200 ml of complex medium in 500 ml serum bottles on acetate and ethanol. A complex medium was used consisting of 0.25 g / L NH ₄ Cl, 0.2 g / L MgSO ₄ x 7 H ₂ O, 0.31 g / L K ₂ HPO ₄ , 0.23 g / L KH ₂ PO ₄ , 2.5 g / l NaHCO ₃ , 1 g / l yeast extract, 10 g / l K-acetate, 20 g / l ethanol, 0.25 g / l L-cysteine-HCl, 1.5 mg / l FeCl ₂ x 4 H ₂ O, 70 μg / L ZnCl ₂ x 7 H ₂ O, 100 μg / L MnCl ₂ x 4 H ₂ O, 6 μg / L Boric Acid, 190 μg / L CoCl ₂ x 6 H ₂ O , 2 μg / L CuCl ₂ x 6 H ₂ O, 24 μg / L NiCl ₂ x 6 H ₂ O, 36 μg / L Na ₂ MoO ₄ x 2 H ₂ O, 3 μg / L Na ₂ SeOO ₃ x 5 H ₂ O, 4 μg / L Na ₂ WO ₄ x 2 H ₂ O, 100 μg / L Vitamin B12, 80 μg / L p-aminobenzoic acid, 20 μg / L Biotin, 200 μg / L Nicotinic acid, 100 μg / L Ca-pantothenic acid, 300 mg / l pyridoxine-HCl, 200 mg / l thiamine-HCl x H ₂ O. The bottle for yvorotki continuously shaken in an open water bath Innova 3100 from New Brunswick Scientific at 37 ° C and shaking speed of 100 min ^-1. Cells were harvested in the late logarithmic phase with OD ₆₀₀ 0.86 and pH 6.01 by anaerobic centrifugation (4500 min ^-1 , 4300 g, 20 ° C, 10 min). The supernatant was removed and the precipitate was again suspended in 10 ml of the above complex medium. This cell suspension was then used for inoculation in a co-culture experiment after 49 hours of passage of the experiment.

During the experiment, 5 ml samples were taken to determine OD ₆₀₀ , pH, and product concentration. The latter was determined by quantitative analysis using ¹ H-NMR spectroscopy.

After inoculation of C. ljungdahlii, cells began to grow and continuously produced acetate to a concentration of ~ 1.3 g / L and ethanol to a concentration of ~ 0.8 g / L after 49 hours. Then, during the process, 49 hours C. kluyveri was inoculated into the reactor. In the further dynamics of the experiment, the production of butyrate and hexanoate was measured to a concentration of 0.6 g / L each. In parallel with the production of butyrate and hexanoate by C. kluyveri, C. ljungdahlii converted butyrate to butanol to a maximum concentration of 472 mg / l butanol and converted hexanoate to hexanol to a maximum concentration of 630 mg / l hexanol.

Example 10

Co-cultivation of Clostridium autoethanogenum and Clostridium kluyveri in a complex medium with CO-containing gas to produce higher alcohols, such as hexanol and octanol (10% CO)

In this example, C. autoethanogenum as the first organism was autotrophically cultured in a complex medium to produce acetate and ethanol. After a predetermined time, C. kluyveri as a second organism was inoculated into the same reactor to convert acetate and ethanol to butyrate and hexanoate. Then, in the next step, C. autoethanogenum converts butyrate to butanol and hexanoate to hexanol. Octanol production was also observed.

Used a complex medium for the joint cultivation of both microorganisms, consisting of 1 g / l NH ₄ Cl, 0.1 g / l KCl, 0.2 g / l MgSO ₄ x 7 H ₂ O, 0.8 g / l NaCl, 0 , 1 g / l KH ₂ PO ₄ , 20 mg / l CaCl ₂ x 2 H ₂ O, 20 g / l MES, 1 g / l yeast extract, 0.4 g / l L-cysteine-HCl, 0.4 g / l Na ₂ S x 9 H ₂ O, 20 mg / l nitrilotriacetic acid, 10 mg / l MnSO ₄ x H ₂ O, 8 mg / l (NH ₄ ) ₂ Fe (SO ₄ ) ₂ x 6 H ₂ O , 2 mg / L CoCl ₂ x 6 H ₂ O, 2 mg / L ZnSO ₄ x 7 H ₂ O, 0.2 mg / L CuCl ₂ x 2 H ₂ O, 0.2 mg / L Na ₂ MoO ₄ x 2 H ₂ O, 0.2 mg / L NiCl ₂ x 6 H ₂ O, 0.2 mg / L Na ₂ SeO ₄ , 0.2 mg / L Na ₂ WO ₄ x 2 H ₂ O, 20 μg / L biotin, 20 g / l of folic acid, 100 micrograms / l pyridoxine-HCl, 50 mg / l thiamine-HCl x H ₂ O, 50 g / l of riboflavin, 50 g / l nicotine oh acid 50 g / L Ca-pantothenic acid, 1 mg / l of vitamin B _12, 50 mcg / l p-aminobenzoic acid, 50 g / l lipoic acid.

Autotrophic cultivation was performed in 500 ml of complex medium in a 1 L serum bottle, which was continuously gassed with synthesis gas consisting of 60% H2, 30% CO2, and 10% CO, at a rate of ~ 1.0 l / h. Gas was introduced into the liquid phase using a microbubble disperser with a pore diameter of 10 μm. The whey bottle was continuously shaken in an Innova 3100 New Brunswick Scientific open water bath at 37 ° C. and a shaking speed of 150 min ⁻¹ . The pH was adjusted once during the experiment by adding anaerobic KOH.

At the beginning of the experiment, C. autethanogenum was inoculated with OD _{600 of} 0.1 autotrophically grown cells. Thus, C. autethanogenum was grown in the complex medium described above under continuous aeration with synthesis gas consisting of 67% H ₂ and 33% CO2 at a rate of 1 l / h in 1 liter serum bottles with 500 ml of complex medium. Gas was introduced into the liquid phase using a microbubble disperser with a pore diameter of 10 μm. The whey bottle was continuously shaken in an Innova 3100 New Brunswick Scientific open water bath at 37 ° C. and a shaking speed of 150 min ⁻¹ . Cells were harvested in the late logarithmic phase with OD ₆₀₀ 0.62 and pH 5.15 by anaerobic centrifugation (4500 min ^-1 , 4300 g, 20 ° C, 10 min). The supernatant was removed and the precipitate was again suspended in 10 ml of the above complex medium. This cell suspension was then used for inoculation in a co-culture experiment. In the late phase of the experiment, C. autethanogenum was again inoculated into an ongoing experiment with OD _{600 of} 0.2 from a pre-culture grown under the same conditions as described above. Cells were harvested in the late logarithmic phase with an OD _{600 of} 0.55 and a pH of 5.12 by anaerobic centrifugation (4500 min ^-1 , 4300 g, 20 ° C, 10 min). The supernatant was removed, and the sediment was again suspended in 10 ml of medium, anaerobically taken from a passing experiment. This cell suspension was then used to transfer the cells back to the co-culture experiment after 74 hours of the experiment.

In parallel, C. kluyveri was grown heterotrophically in 200 ml of complex medium in 500 ml serum bottles on acetate and ethanol. A complex medium was used consisting of 0.25 g / L NH ₄ Cl, 0.2 g / L MgSO ₄ x 7 H ₂ O, 0.31 g / L K ₂ HPO ₄ , 0.23 g / L KH ₂ PO ₄ , 2.5 g / l NaHCO ₃ , 1 g / l yeast extract, 10 g / l K-acetate, 20 g / l ethanol, 0.25 g / l L-cysteine-HCl, 1.5 mg / l FeCl ₂ x 4 H ₂ O, 70 μg / L ZnCl ₂ x 7 H ₂ O, 100 μg / L MnCl ₂ x 4 H ₂ O, 6 μg / L Boric Acid, 190 μg / L CoCl ₂ x 6 H ₂ O , 2 μg / L CuCl ₂ x 6 H ₂ O, 24 μg / L NiCl ₂ x 6 H ₂ O, 36 μg / L Na ₂ MoO ₄ x 2 H ₂ O, 3 μg / L Na ₂ SeOO ₃ x 5 H ₂ O, 4 μg / L Na ₂ WO ₄ x 2 H ₂ O, 100 μg / L Vitamin B12, 80 μg / L p-aminobenzoic acid, 20 μg / L Biotin, 200 μg / L Nicotinic acid, 100 μg / L Ca-pantothenic acid, 300 mg / l pyridoxine-HCl, 200 mg / l thiamine-HCl x H ₂ O. The bottle for yvorotki continuously shaken in an open water bath Innova 3100 from New Brunswick Scientific at 37 ° C and shaking speed of 100 min ^-1. Cells were harvested in the late logarithmic phase with OD ₆₀₀ 0.86 and pH 6.01 by anaerobic centrifugation (4500 min ^-1 , 4300 g, 20 ° C, 10 min). The supernatant was removed, and the sediment was again suspended in 10 ml of medium anaerobically taken from their ongoing experiment. This cell suspension was then used to transfer the cells to the co-culture experiment after 74 hours of the experiment. In the late phase of the experiment, C. kluyveri was again inoculated into the ongoing experiment with an OD _{600 of} 0.15 from a pre-culture grown under the same conditions as described above. Cells were harvested in a logarithmic phase with OD ₆₀₀ 0.38 and pH 6.67 by anaerobic centrifugation (4500 min ^-1 , 4300 g, 20 ° C, 10 min). The supernatant was removed, and the sediment was again suspended in 10 ml of medium, anaerobically taken from a passing experiment. This cell suspension was then used to transfer the cells back to the co-culture experiment after 74 hours of the experiment.

During the experiment, 5 ml samples were taken to determine OD ₆₀₀ , pH, and product concentration. The latter was determined by quantitative analysis using ¹ H-NMR spectroscopy.

After inoculation of C. autoethanogenum, the cells began to grow and produced acetate to a concentration of ~ 2.2 g / L to a concentration of ~ 0.5 g / L after 23 hours. At this point in time, C. kluyveri was inoculated into an ongoing experiment and subsequently received butyrate and hexanoate. Butyrate and hexanoate were then reduced to the corresponding alcohols using C. autoethanogenum. After 69 hours of the experiment, the pH was raised from pH 4.74 to pH 6.01 by adding anaerobic KOH. After that, 50 mg / L L-cysteine HCl was added to the medium and both C. autoethanogenum and C. kluyveri cells were inoculated into the ongoing experiments. After 140 hours of cultivation, 650 mg / L of butanol, 220 mg / L of hexanol and 4.5 mg / L of octanol were obtained.

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Claims (20)

1. The reaction mixture to produce at least one higher alcohol in an aqueous medium, containing a mixed culture of the first and second microorganisms in an aqueous medium containing gaseous carbon monoxide, where - the first microorganism is an acetogenic microorganism capable of converting a carbon source into acetate and / or ethanol, and is selected from Clostridium ljungdahlii and Clostridium autoethanogenum , and - the second microorganism capable of converting acetate and / or ethanol to form acid is Clostridium kluyveri , where the first microorganism is additionally able to convert the acid into the corresponding higher alcohol, and the higher alcohol contains at least 6 carbon atoms. 2. The mixture of claim 1, wherein the first microorganism is Clostridium ljungdahlii . 3. The mixture according to claim 1, where the higher alcohol is a C ₆ -C ₈ alcohol. 4. The mixture according to claim 1, where the carbon source contains at least 2% by volume of gaseous carbon monoxide relative to the volume of the carbon source. 5. The mixture according to claim 1, where the carbon source contains 7% -65% by volume of gaseous carbon monoxide. 6. The mixture of claim 1, wherein the aqueous medium further comprises free oxygen. 7. The mixture according to claim 6, where the mixture contains acetogenic bacteria in the logarithmic phase and acetogenic bacteria in the stationary phase. 8. The mixture according to claim 7, where the first acetogenic microorganism in the exponential phase of growth is characterized by a growth rate of not more than 0.062 h ^-1 . 9. The mixture according to claim 7, where the first acetogenic microorganism in the exponential growth phase is characterized by OD ₆₀₀ from 0.01 to 1.95. 10. A method of obtaining at least one higher alcohol in an aqueous medium containing a reaction mixture containing a first and second microorganism, where - the first microorganism is an acetogenic microorganism capable of converting a carbon source containing CO into acetate and / or ethanol, and is selected from Clostridium ljungdahlii and Clostridium autoethanogenum , and - the second microorganism capable of converting acetate and / or ethanol to form acid is Clostridium kluyveri , where the first microorganism is additionally capable of converting the acid into the corresponding higher alcohol containing at least 6 carbon atoms. 11. The method according to p. 10, where the carbon source contains 7% -65% by volume of gaseous carbon monoxide. 12. The method according to p. 10, where the higher alcohol is selected from the group consisting of 1-hexanol, 1-octanol and 1-heptanol. 13. The method according to p. 10, where the reaction mixture corresponds to p. 1. 14. The use of the reaction mixture according to p. 1 to obtain at least one higher alcohol containing at least 6 carbon atoms.